Methods for the diagnosis of body weight disorders including obesity

ABSTRACT

The present invention relates to methods and compositions for the treatment of body weight disorders, including, but not limited to, obesity. Specifically, the present invention identifies and describes genes which are differentially expressed in body weight disorder states, relative to their expression in normal, or non-body weight disorder states, and/or in response to manipulations relevant to appetite and/or weight regulation. Further, the present invention identifies and describes genes via the ability of their gene products to interact with gene products involved in body weight disorders and/or appetite and/or body weight regulation. Still further, the present invention provides methods for the identification and therapeutic use of compounds as treatments of body weight disorders. Additionally, the present invention describes methods for the diagnostic evaluation and prognosis of various body weight disorders, and for the identification of subjects exhibiting a predisposition to such conditions.

This is a continuation-in-part of Ser. No. 08/470,868, filed Jun. 6,1995, which is a continuation-in-part of Ser. No. 08/294,522, filed Aug.23, 1994, both applications of which are incorporated herein byreference in their entirety.

1. INTRODUCTION

The present invention relates to methods and compositions for thetreatment of body weight disorders, including, but not limited to,obesity. Specifically, the present invention identifies and describesgenes which are differentially expressed in body weight disorder states,relative to their expression in normal, or non-body weight disorderstates, and also identifies genes which are differentially expressed inresponse to manipulations relevant to appetite and/or weight regulation.Further, the present invention identifies and describes genes via theability of their gene products to interact with gene products involvedin body weight disorders and/or to interact with gene products which arerelevant to appetite and/or body weight regulation. Still further, thepresent invention provides methods for the identification andtherapeutic use of compounds as treatments of body weight disorders.Additionally, the present invention describes methods for the diagnosticevaluation and prognosis of various body weight disorders, and for theidentification of subjects exhibiting a predisposition to suchconditions.

2. BACKGROUND OF THE INVENTION

Body weight disorders, including eating disorders, represent majorhealth problems in all industrialized countries. Obesity, the mostprevalent of eating disorders, for example, is the most importantnutritional disorder in the western world, with estimates of itsprevalence ranging from 30% to 50% within the middle-aged population.Other body weight disorders, such as anorexia nervosa and bulimianervosa which together affect approximately 0.2% of the femalepopulation of the western world, also pose serious health threats.Further, such disorders as anorexia and cachexia (wasting) are alsoprominent features of other diseases such as cancer, cystic fibrosis,and AIDS.

Obesity, defined as an excess of body fat relative to lean body mass,also contributes to other diseases. For example, this disorder isresponsible for increased incidences of diseases such as coronary arterydisease, stroke, and diabetes. Obesity is not merely a behavioralproblem, i.e., the result of voluntary hyperphagia. Rather, thedifferential body composition observed between obese and normal subjectsresults from differences in both metabolism and neurologic/metabolicinteractions. These differences seem to be, to some extent, due todifferences in gene expression, and/or level of gene products oractivity. The nature, however, of the genetic factors which control bodycomposition are unknown, and attempts to identify molecules involved insuch control have generally been empiric and the parameters of bodycomposition and/or substrate flux are monitored have not yet beenidentified (Friedman, J. M. et al., 1991, Mammalian Gene 1:130-144).

The epidemiology of obesity strongly shows that the disorder exhibitsinherited characteristics, (Stunkard, 1990, N. Eng. J. Med. 322:1483).Moll et al., have reported that, in many populations, obesity seems tobe controlled by a few genetic loci, (Moll et al. 1991, Am. J. Hum. Gen.49:1243). In addition, human twin studies strongly suggest a substantialgenetic basis in the control of body weight, with estimates ofheritability of 80-90% (Simopoulos, A. P. & Childs B., eds., 1989, in"Genetic Variation and Nutrition in Obesity", World Review of Nutritionand Diabetes 63, S. Karger, Basel, Switzerland; Borjeson, M., 1976,Acta. Paediatr. Scand. 65:279-287).

Further, studies of non-obese persons who deliberately attempted to gainweight by systematically over-eating were found to be more resistant tosuch weight gain and able to maintain an elevated weight only by veryhigh caloric intake. In contrast, spontaneously obese individuals areable to maintain their status with normal or only moderately elevatedcaloric intake.

In addition, it is a commonplace experience in animal husbandry thatdifferent strains of swine, cattle, etc., have different predispositionsto obesity. Studies of the genetics of human obesity and of models ofanimal obesity demonstrate that obesity results from complex defectiveregulation of both food intake, food induced energy expenditure and ofthe balance between lipid and lean body anabolism.

There are a number of genetic diseases in man and other species whichfeature obesity among their more prominent symptoms, along with,frequently, dysmorphic features and mental retardation. Although nomammalian gene associated with an obesity syndrome has yet beencharacterized in molecular terms, a number of such diseases exist inhumans. For example, Prader-Willi syndrome (PWS) affects approximately 1in 20,000 live births, and involves poor neonatal muscle tone, facialand genital deformities, and generally obesity. The genetics of PWS arevery complex, involving, for example, genetic imprinting, in whichdevelopment of the disease seems to depend upon which parent contributesthe abnormal PWS allele. In approximately half of all PWS patients,however, a deletion on the long arm of chromosome 11 is visible, makingthe imprinting aspect of the disease difficult to reconcile. Given thevarious symptoms generated, it seems likely that the PWS gene productmay be required for normal brain function, and may, therefore, not bedirectly involved in adipose tissue metabolism.

In addition to PWS, many other pleiotropic syndromes which includeobesity as a symptom have been characterized. These syndromes are moregenetically straightforward, and appear to involve autosomal recessivealleles. The diseases, which include, among others, Ahlstroem,Carpenter, Bardet-Biedl, Cohen, and Morgagni-Stewart-Monel Syndromes.

Animals having mutations which lead to syndromes that include obesitysymptoms have also been identified. Attempts have been made to utilizesuch animals as models for the study of obesity. The best studied animalmodels for genetic obesity are mice which contain the autosomalrecessive mutations ob/ob (obese) and db/db (diabetes). These mutationsare on chromosomes 6 and 4, respectively, but lead to clinically similarpictures of obesity, evident starting at about 1 month of age, whichinclude hyperphagia, severe abnormalities in glucose and insulinmetabolism, very poor thermo-regulation and non-shivering thermogenesis,and extreme torpor and underdevelopment of the lean body mass.Restriction of the diet of these animals to restore a more normal bodyfat mass to lean body mass ration is fatal and does not result in anormal habitus.

Although the phenotypes of db/db and ob/ob mice are similar, the lesionsare distinguishable by means of parabiosis. The feeding of normal miceand, putatively, all mammals, is regulated by satiety factors. The ob/obmice are apparently unable to express the satiety factor, while thedb/db mouse is unresponsive to it.

In addition to ob and db, several other single gene mutations resultingin obesity in mice have been identified. These include the yellowmutation at the agouti locus, which causes a pleiotropic syndrome whichcauses moderate adult onset obesity, a yellow coat color, and a highincidence of tumor formation (Herberg, L. and Coleman, D. L., 1977,Metabolism 26:59), and an abnormal anatomic distribution of body fat(Coleman, D. L., 1978, Diabetologia 14:141-148). Additionally, mutationsat the fat and tubby loci cause moderately severe, maturity-onsetobesity with somewhat milder abnormalities in glucose homeostasis thanare observed in ob and db mice (Coleman, D. L., and Eicher, E. M., 1990,J. Heredity 81:424-427). Further, autosomal dominant mutations at theadipose locus of chromosome 7, have been shown to cause obesity.

Other animal models include fa/fa (fatty) rats, which bear manysimilarities to the ob/ob and db/db mice, discussed above. Onedifference is that, while fa/fa rats are very sensitive to cold, theircapacity for non-shivering thermogenesis is normal. Torpor seems to playa larger part in the maintenance of obesity in fa/fa rats than in themice mutants. In addition, inbred mouse strains such as NZO mice andJapanese KK mice are moderately obese. Certain hybrid mice, such as theWellesley mouse, become spontaneously fat. Further, several desertrodents, such as the spiny mouse, do not become obese in their naturalhabitats, but do become so when fed on standard laboratory feed.

Animals which have been used as models for obesity have also beendeveloped via physical or pharmacological methods. For example,bilateral lesions in the ventromedial hypothalamus (VMH) andventrolateral hypothalamus (VLH) in the rat are associated,respectively, with hyperphagia and gross obesity and with aphagia andcachexia. Further, it has been demonstrated that feedingmonosodium-glutamate (MSG) to new born mice also results in an obesitysyndrome.

Attempts have been made to utilize such animal models in the studymolecular causes of obesity. For example, adipsin, a murine serineprotease with activity closely similar to human complement factor D,produced by adipocytes, has been found to be suppressed in ob/ob, db/dband MSG-induced obesity (Flier, 1987, Science 237:405). The suppressionof adipsin precedes the onset of obesity in each model (Lowell, 1990,Endocrinology 126:1514). Further studies have mapped the locus of thedefect in these models to activity of the adipsin promoter (Platt, 1989,Proc. Natl. Acad. Sci. USA 86:7490). Further, alterations have beenfound in the expression of neuro-transmitter peptides in thehypothalamus of the ob/ob mouse (Wilding, 1993, Endocrinology 132:1939),of glucose transporter proteins in islet β-cells (Ohneda, 1993, Diabetes42:1065) and of the levels of G-proteins (McFarlane-Anderson, 1992,Biochem. J. 282:15).

To date however, no gene, in humans, has been found which is causativein the processes leading to obesity. Given the severity and prevalence,however, of disorders, including obesity, which affect body weight andbody composition, there exists a great need for the systematicidentification of such body weight disorder-causing genes.

3. SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for thetreatment of body weight disorders, including, but not limited to,obesity. Specifically, the present invention identifies and describesgenes which are differentially expressed in body weight disorder states,relative to their expression in normal, or non-body weight disorderstates, and also identifies genes which are differentially expressed inresponse to manipulations relevant to appetite and/or body weightregulation. Such differentially expressed genes may represent "targetgenes" and/or "fingerprint genes". Further, the present inventionidentifies and describes genes, termed "pathway genes", via the abilityof their gene products to interact with gene products involved in bodyweight disorders and/or to interact with gene products which arerelevant to appetite and body weight regulation. Pathway genes may alsoexhibit target gene and/or fingerprint gene characteristics.

"Differential expression", as used herein, refers to both quantitativeas well as qualitative differences in the genes' temporal and/or tissueexpression patterns. "Fingerprint gene," as used herein, refers to adifferentially expressed gene whose expression pattern may be utilizedas part of a prognostic or diagnostic body weight disorder evaluation,or which, alternatively, may be used in methods for identifyingcompounds useful for the treatment of body weight disorders. "Targetgene", as used herein, refers to a differentially expressed geneinvolved in body weight disorders and/or appetite or body regulationsuch that modulation of the level of target gene expression or of targetgene product activity may act to ameliorate symptoms of body weightdisorders including, but are not limited to, obesity.

This invention is based, in part on systematic, search strategiesinvolving body weight disorder experimental paradigms coupled withsensitive gene expression assays.

The present invention also describes the products of such fingerprint,target, and pathway genes, describes antibodies to such gene products,and still further describes cell- and animal-based models of body weightdisorders to which such gene products may contribute.

The invention further provides methods for the identification ofcompounds which modulate the expression of genes or the activity of geneproducts involved in body weight disorders and processes relevant toappetite and/or body weight regulation. Still further, the presentinvention describes methods for the treatment of body weight disorderswhich may involve the administration of such compounds to individualsexhibiting body weight disorder symptoms or tendencies.

Additionally, the present invention describes methods for prognostic anddiagnostic evaluation of various body weight disorders, and for theidentification of subjects exhibiting a predisposition to suchdisorders.

The Examples presented in Sections 6-10, below, demonstrate thesuccessful use of the body weight disorder paradigms of the invention toidentify body weight disorder target genes.

4. DESCRIPTION OF THE FIGURES

FIG. 1. Differential display comparing RNAs from liver tissue of leanand obese mice. Each group (1-11) of four lanes shows the patternobtained with one primer pair combination, for a total of elevendifferent primer pair combinations. All lanes are products of apolymerase chain reaction (PCR) in which T₁₁ GG was used as the 3'oligonucleotide and one of eleven different arbitrary 10 meroligonucleotides was used as the 5' oligonucleotide. Within each groupof four lanes, the loading is as follows, from left to right: C57B1/6Jlean control (marked "C"); C57B1/6J ob/ob (marked "ob"); C57B1/Ks leancontrol (marked "C"); and C57B1/Ks db/db (marked "db"). An arrowindicates a band (designated L36) that is differential between obese andlean samples amplified by the same primer pair, specifically, primerpair 6.

FIG. 2. Northern blot analysis confirming differential regulation of agene corresponding to band L36. Poly A⁺ RNA (1 μg/lane) obtained fromthe original liver total RNA preparations was hybridized with a cDNAprobe prepared by random priming of reamplified lane L36 (see materialsand methods, below, in Section 6.1). Lane 1, C57B1/6J lean control("C"); lane 2, C57B1/6J ob/ob ("ob"); lane 3 C57B1/Ks lean control("C"); and lane 4, C57B1/Ks db/db ("db").

FIG. 3A. Consensus nucleotide sequence of L36 amplified band (SEQ. IDNO: 1). The IUPAC-IUB Standard Code is used (with the addition of the"X" designation, as shown below) in this nucleotide sequence and thosenucleotide sequences listed in the figures which follow. Upper caseletters refer to perfect consensus matches at a particular base pairposition. Lower case letters refer to base pair positions at which therewas a less than perfect consensus match. Specifically, the code used wasas follows:

    ______________________________________                                        Code        Base           Meaning                                            ______________________________________                                        A           A              Adenine                                            C           C              Cytosine                                           G           G              Guanine                                            T           T              Thymine                                            U           U              Uracil (RNA)                                       R           A or G         Purine                                             Y           C or T (or U)  Pyrimidine                                         K           G or T (or U)  Keto                                               M           A or C         Amino                                              S           G or C         Strong                                             W           A or T (or U)  Weak                                               B           C, G, T (or U) not A                                              D           G, A, T (or U) not C                                              H           A, C, T (or U) not G                                              V           A, C, or G     not T                                              N           A, C, G, T     Any                                                            (or U)                                                            X           A, C, G, T     Any or none                                                    (or U), or none                                                   ______________________________________                                    

FIG. 3B. Alignment of L36 consensus nucleotide sequence with a mousestearoyl-CoA desaturase nucleotide sequence (top sequence: SEQ ID NO:2;bottom sequence: SEQ ID NO:3). It will be noted that there are twoalignments listed for this L36/mouse stearoyl-CoA desaturase match. Eachrepresents a highly statistically significant alignment, and together,these alignments represent very highly significant matches. This is thecase for each of the matches listed in the figures, below, which havegreater than one alignment listed.

FIG. 4. Alignment of P3 consensus nucleotide sequence (top line of uppersequence pair: SEQ ID NO.:4; top line of lower sequence pair: SEQ IDNO.:39) with a mouse glutamine synthetase nucleotide sequence (bottomline of upper sequence pair: SEQ ID NO.:5; bottom line of lower sequencepair: SEQ ID NO.:6).

FIG. 5. Alignment of P13 consensus nucleotide sequence (top line offirst sequence pair: SEQ ID NO.:7; top line of second sequence pair: SEQID NO.:40; top line of third sequence pair: SEQ ID NO.:41; top line offourth sequence pair: SEQ ID NO.:42) with a mouse islet regeneratingprotein nucleotide sequence (bottom line of first sequence pair: SEQ IDNO.:8; bottom line of second sequence pair: SEQ ID NO.:9; bottom line ofthird sequence pair: SEQ ID NO.:10; bottom line of fourth sequence pair:SEQ ID NO.:11).

FIG. 6. Alignment of F5 consensus nucleotide sequence (top line ofsequence pair: SEQ ID NO.:12) with a mouse alpha-amylase nucleotidesequence (bottom line of sequence pair: SEQ ID NO.:13).

FIG. 7. Alignment of murine C5 consensus nucleotide sequence (top lineof sequence pair: SEQ ID NO.:14) with a rabbit uncoupling proteinnucleotide sequence (bottom line of sequence pair: SEQ ID NO.:15).

FIGS. 8A, 8B. Alignment of L31/F74 consensus nucleotide sequence (topline of sequence pair; SEQ ID NO.:16) with a mouse major urinary proteinII nucleotide sequence (bottom line of sequence pair; SEQ ID NO.:17).

FIG. 9. Alignment of L7/L21 consensus nucleotide sequence (top line ofsequence pair; SEQ ID NO.:18) with a mouse cytochrome oxidase c subunitI nucleotide sequence (bottom line of sequence pair; SEQ ID NO.:19).

FIG. 10. Alignment of L29 consensus nucleotide sequence (top line ofsequence pair; SEQ ID NO.:20) with a mouse testosterone 15-alphahydroxylase nucleotide sequence (bottom line of sequence pair; SEQ IDNO.:21).

FIG. 11. Alignment of L38 consensus nucleotide sequence (top line ofupper sequence pair: SEQ ID NO.:22; top line of lower sequence pair: SEQID NO.:43) with a mouse 24p3 (a lipocalin family member of unknownfunction) nucleotide sequence (bottom line of upper sequence pair: SEQID NO.:23; bottom line of lower sequence pair: SEQ ID NO.:24).

FIG. 12. Alignment of L37 consensus nucleotide sequence (top line offirst sequence pair; SEQ ID NO.:25; top line of second sequence pair:SEQ ID NO.:44; top line of third sequence pair: SEQ ID NO.:45) with amouse p6-5 (a mouse sequence 86% homologous to rat preproelastase I)nucleotide sequence (bottom line of first sequence pair: SEQ ID NO.:26;bottom line of second sequence pair: SEQ ID NO.:27; bottom line of thirdsequence pair: SEQ ID NO.:28).

FIG. 13. Alignment of L57 consensus nucleotide sequence (top line offirst sequence pair: SEQ ID NO.:29; top line of second sequence pair:SEQ ID NO.:46; top line of third sequence pair: SEQ ID NO.:47; top lineof fourth sequence pair: SEQ ID NO.:48) with a mouse orphan hormonereceptor nucleotide sequence (bottom line of first sequence pair: SEQ IDNO.:30; bottom line of second sequence pair: SEQ ID NO.;31; bottom lineof third sequence pair: SEQ ID NO.:32; bottom line of fourth sequencepair: SEQ ID NO.:33).

FIG. 14. Full length F49 cDNA clone. A cDNA putatively encoding the fulllength coding sequence of the F49 gene was isolated and its nucleotidesequence is listed herein (SEQ ID NO.:34). The F49 coding sequenceencodes a 96 amino acid protein whose sequence is also listed herein(SEQ ID NO.:35). The initiating methionine codon and the terminationcodon are boxed.

FIG. 15. Hydropathy plot of the F49 gene product.

FIGS. 16A, 16B. Depicted herein are, first, the full length mouse C5nucleotide sequence (SEQ ID NO.:36), and second, the amino acid sequence(SEQ ID NO.:37) encoded by the mouse C5 gene. The initiating methionineand the termination codon are boxed.

FIGS. 17A, 17B. Human C5 cDNA. The bottom line of the Figure depicts thenucleotide sequence of a human C5 cDNA clone (SEQ ID NO.:38); the topline of the Figure depicts the portion of the human C5 amino acidsequence (SEQ ID NO: 56) derived from the cDNA nucleotide sequence.

FIG. 18. Full length C5 amino acid sequence (SEQ ID NO: 51).

FIG. 19. Northern analysis of tissue distribution of human C5 mRNA.

FIG. 20. Alignment of H27 consensus nucleotide sequence (top line ofsequence pair; SEQ ID NO: 49) with a mouse autoantigen La nucleotidesequence (SEQ ID NO: 50).

FIG. 21. Alignment of F84 consensus nucleotide sequence (top line ofupper sequence pair: SEQ ID NO.:52; top line of lower sequence pair: SEQID NO.:54) with a mouse cytochrome p450 IID nucleotide sequence (bottomline of upper sequence pair: SEQ ID NO.:53; bottom line of lowersequence pair: SEQ ID NO.:55).

FIG. 22. Mouse L34 cDNA nucleotide sequence (SEQ ID NO: 57).

5. DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the treatment of body weight disorders,including, but not limited to, obesity. Genes, termed "target genes"and/or "fingerprint genes", are described which are differentiallyexpressed in body weight disorder states, relative to their expressionin normal, or non-body weight disorder states, and/or which aredifferentially expressed in response to manipulations relevant toappetite and/or body weight regulation. Additionally, genes, termed"pathway genes", are described whose gene products exhibit an ability tointeract with gene products involved in body weight disorders and/orwith gene products which are relevant to appetite and/or body weightregulation. Methods for the identification of such fingerprint, target,and pathway genes are also described.

Further, the gene products of such fingerprint, target, and pathwaygenes are described, antibodies to such gene products are described, asare cell- and animal-based models of body weight disorders to which suchgene products may contribute.

Described, below, are methods for the identification of compounds whichmodulate the expression of genes or the activity of gene productsinvolved in body weight disorders and processes relevant to appetiteand/or body weight regulation. Additionally described, below, aremethods for the treatment of body weight disorders.

Also discussed, below, are methods for prognostic and diagnosticevaluation of various body weight disorders, and for the identificationof subjects exhibiting a predisposition to such disorders.

5.1. Identification of Differentially Expressed and Pathway Genes

Described herein are methods for the identification of genes which areinvolved in body weight disorder states, and/or which are involved inappetite and body weight regulation. Such genes may represent geneswhich are differentially expressed in body weight disorder statesrelative to their expression in normal, or non-body weight disorderstates. Further, such genes may represent genes which are differentiallyregulated in response to manipulations relevant to appetite and bodyweight regulation. Such differentially expressed genes may represent"target" and/or "fingerprint" genes. Methods for the identification ofsuch differentially expressed genes are described, below, in Section5.1.1. Methods for the further characterization of such differentiallyexpressed genes, and for their identification as target and/orfingerprint genes, are presented, below, in Section 5.1.3.

In addition, methods are described herein, in Section 5.1.2, for theidentification of genes, termed "pathway genes", involved in body weightdisorder states, and/or in appetite or body weight regulation. "Pathwaygene", as used herein, refers to a gene whose gene product exhibits theability to interact with gene products involved in body weight disordersand/or to interact with gene products which are relevant to appetite orbody weight regulation. A pathway gene may be differentially expressedand, therefore, may have the characteristics of a target and/orfingerprint gene.

"Differential expression" as used herein refers to both quantitative aswell as qualitative differences in the genes' temporal and/or tissueexpression patterns. Thus, a differentially expressed gene mayqualitatively have its expression activated or completely inactivated innormal versus body weight disorder states, or under control versusexperimental conditions. Such a qualitatively regulated gene willexhibit an expression pattern within a given tissue or cell type whichis detectable in either control or body weight disorder subjects, but isnot detectable in both. Alternatively, such a qualitatively regulatedgene will exhibit an expression pattern within a given tissue or celltype which is detectable in either control or experimental subjects, butis not detectable in both. "Detectable", as used herein, refers to anRNA expression pattern which is detectable via the standard techniquesof differential display, RT-PCR and/or Northern analyses, which are wellknown to those of skill in the art.

Alternatively, a differentially expressed gene may have its expressionmodulated, i.e., quantitatively increased or decreased, in normal versusbody weight disorder states, or under control versus experimentalconditions. The degree to which expression differs in normal versus bodyweight disorder or control versus experimental states need only be largeenough to be visualized via standard characterization techniques, suchas, for example, the differential display technique described below.Other such standard characterization techniques by which expressiondifferences may be visualized include but are not limited to,quantitative RT (reverse transcriptase) PCR and Northern analyses.

Differentially expressed genes may be further described as target genesand/or fingerprint genes. "Fingerprint gene," as used herein, refers toa differentially expressed gene whose expression pattern may be utilizedas part of a prognostic or diagnostic body weight disorder evaluation,or which, alternatively, may be used in methods for identifyingcompounds useful for the treatment of body weight disorders. Afingerprint gene may also have the characteristics of a target gene or apathway gene.

"Target gene", as used herein, refers to a differentially expressed geneinvolved in body weight disorders and/or appetite or body regulation ina manner by which modulation of the level of target gene expression orof target gene product activity may act to ameliorate symptoms of bodyweight disorders including, but are not limited to, obesity. A targetgene may also have the characteristics of a fingerprint gene and/or apathway gene.

5.1.1. Methods for the Identification of Differentially Expressed Genes

A variety of methods may be utilized for the identification of geneswhich are involved in body weight disorder states, and/or which areinvolved in appetite and body weight regulation. Described in Section5.1.1.1 are several experimental paradigms which may be utilized for thegeneration of subjects and samples which may be used for theidentification of such genes. Material from the paradigm control andexperimental subjects may be characterized for the presence ofdifferentially expressed gene sequences as discussed, below, in Section5.1.1.2.

5.1.1.1. Paradigms for the Identification of Differentially ExpressedGenes

Among the paradigms which may be utilized for the identification ofdifferentially expressed genes involved in, for example, body weightdisorders, are paradigms designed to analyze those genes which may beinvolved in short term appetite control. Accordingly, such paradigms arereferred to as "short term appetite control paradigms." These paradigmsmay serve to identify genes involved in signalling hunger and satiety.

In one embodiment of such a paradigm, test subjects, preferably mice,may be fed normally prior to the initiation of the paradigm study, thendivided into one control and two experimental groups. The control groupwould then be maintained on ad lib nourishment, while the firstexperimental group ("fasted group") would be fasted, and the secondexperimental group ("fasted-refed group") would initially be fasted, andwould then be offered a highly palatable meal shortly before thecollection of tissue samples. Each test animal should be weightedimmediately prior to and immediately after the experiment. The Examplepresented in Section 7, below, demonstrates the use of such short termappetite paradigms to identify gene sequences which are differentiallyexpressed in control versus fasting and versus refed animals.

Among additional paradigms which may be utilized for the identificationof differentially expressed genes involved in, for example, body weightdisorders, are paradigms designed to analyze those genes which may beinvolved genetic obesity. Accordingly, such paradigms are referred to as"genetic obesity paradigms". In the case of mice, for example, suchparadigms may identify genes regulated by the ob, db, and/or tub geneproducts. In the case of rats, for example, such paradigms may identifygenes regulated by the fatty (fa) gene product.

In one embodiment of such a paradigm, test subjects may include ob/ob,db/db, and/or tub/tub experimental mice and lean littermate controlanimals. Such animals would be offered normal nourishment for a givenperiod, after which tissue samples would be collected for analysis. TheExamples presented in Sections 6 and 8, below, demonstrate the use ofsuch genetic obesity paradigms in identifying gene sequences which aredifferentially expressed in obese versus lean animals.

In additional embodiments, ob/ob, db/db, and/or tub/tub experimentalmice and lean control animals may be utilized as part of the short termappetite control paradigms discussed above, or as part of the set pointand/or drug study paradigms discussed below.

Paradigms which may be utilized for the identification of differentiallyexpressed genes involved in body weight disorders may include paradigmsdesigned to identify those genes which may be regulated in response tochanges in body weight. Such paradigms may be referred to as "set pointparadigms".

In one embodiment of such a paradigm, test subjects, preferably mice,may be fed normally prior to the initiation of the paradigm study, thendivided into one control and two experimental groups. The control groupwould then be maintained on an ad lib diet of normal nourishment inorder to calculate daily food intake. The first experimental group("underweight group") would then be underfed by receiving some fractionof normal food intake, 60-90% of normal, for example, so as to reduceand maintain the group's body weight to some percentage, for example80%, of the control group. The second experimental group ("overweightgroup") would be overfed by receiving a diet which would bring the groupto some level above that of the control, for example 125% of the controlgroup. Tissue samples would then be obtained for analysis. The Examplepresented in Section 9, below, demonstrates the use of such set pointparadigms to identify gene sequences which are differentially expressedin control versus overweight and/or underweight conditions.

Additionally, human subjects may be utilized for the identification ofobesity-associated genes. In one embodiment of such a paradigm, tissuesamples may be obtained from obese and lean human subjects and analyzedfor the presence of genes which are differentially expressed in thetissue of one group as opposed to another (e.g. differentially expressedin lean versus obese subjects). In another embodiment, obese humansubjects may be studied over the course of a period of weight loss,achieved through food restriction. Tissue from these previously obesesubjects may be analyzed for differential expression of gene productsrelative to tissue obtained from control (lean, non-previously obese)and obese subjects.

Paradigms may be utilized for the identification of differentiallyexpressed genes involved in body weight disorders may additionallyinclude paradigms designed to identify genes associated with body weightdisorders induced by some physical manipulation to the test subject,such as, for example, hypothalamic lesion-induced body weight disorders.For example, bilateral lesions in the ventromedial hypothalamus (VMH) ofrodents may be utilized to induce hyperphagia and gross obesity in testsubjects, while bilateral lesions in the ventrolateral hypothalamus(VLH) of rodents may be utilized to induce aphagia in test subjects. Insuch paradigms, tissue from hypothalamic-lesioned test subjects and fromcontrol subjects would be analyzed for the identification of genes whichare differentially expressed in control versus lesioned animals.

Drugs known to affect (e.g., ameliorate) human or animal body weightand/or appetite (such as short term appetite) may be incorporated intoparadigms designed to identify genes which are involved in body weightdisorders and/or body weight or appetite regulation. Accordingly, suchparadigms are referred to as "drug study paradigms". Such compounds mayinclude known therapeutics, as well as compounds that are not useful astherapeutics due to, for example, their harmful side effects. Among thecategories of control and test subjects which may be utilized in suchparadigms are, for example, lean subjects, obese subjects, and obesesubjects which have received the drug of interest. In variousembodiments of the paradigms, subjects such as these may be fed a normalad lib diet, a caloric restriction maintained diet, or a caloricrestriction ad lib diet. Control and test subjects may additionally bepairfed i.e., the control and test subjects may be fed via a coupledfeeding device such that both control and test subjects receiveidentical amounts and types of food).

5.1.1.2. Analysis of Paradigm Material

In order to identify differentially expressed genes, RNA, either totalor mRNA, may be isolated from one or more tissues of the subjectsutilized in paradigms such as those described, above, in Section 5.1.1.RNA samples are obtained from tissues of experimental subjects and fromcorresponding tissues of control subjects. Any RNA isolation techniquewhich does not select against the isolation of mRNA may be utilized forthe purification of such RNA samples. See, for example, Ausubel, F. M.et al., eds., 1987-1993, Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. New York, which is incorporated herein by referencein its entirety. Additionally, large numbers of tissue samples mayreadily be processed using techniques well known to those of skill inthe art, such as, for example, the single-step RNA isolation process ofChomczynski, P. (1989, U.S. Pat. No. 4,843,155), which is incorporatedherein by reference in its entirety.

Transcripts within the collected RNA samples which represent RNAproduced by differentially expressed genes may be identified byutilizing a variety of methods which are well known to those of skill inthe art. For example, differential screening (Tedder, T. F. et al.,1988, Proc. Natl. Acad. Sci. USA 85:208-212), subtractive hybridization(Hedrick, S. M. et al., 1984, Nature 308:149-153; Lee, S. W. et al.,1984, Proc. Natl. Acad. Sci. USA 88:2825), and, preferably, differentialdisplay (Liang, P. and Pardee, A. B., 1992, Science 257:967-971; U.S.Pat. No. 5,262,311, which is incorporated herein by reference in itsentirety), may be utilized to identify nucleic acid sequences derivedfrom genes that are differentially expressed.

Differential screening involves the duplicate screening of a cDNAlibrary in which one copy of the library is screened with a total cellcDNA probe corresponding to the mRNA population of one cell type while aduplicate copy of the cDNA library is screened with a total cDNA probecorresponding to the mRNA population of a second cell type. For example,one cDNA probe may correspond to a total cell cDNA probe of a cell typeor tissue derived from a control subject, while the second cDNA probemay correspond to a total cell cDNA probe of the same cell type ortissue derived from an experimental subject. Those clones whichhybridize to one probe but not to the other potentially represent clonesderived from genes differentially expressed in the cell type of interestin control versus experimental subjects.

Subtractive hybridization techniques generally involve the isolation ofmRNA taken from two different sources, e.g., control and experimentaltissue or cell type, the hybridization of the mRNA or single-strandedcDNA reverse-transcribed from the isolated mRNA, and the removal of allhybridized, and therefore double-stranded, sequences. The remainingnon-hybridized, single-stranded cDNAs, potentially represent clonesderived from genes that are differentially expressed in the two mRNAsources. Such single-stranded cDNAs are then used as the startingmaterial for the construction of a library comprising clones derivedfrom differentially expressed genes.

The differential display technique describes a procedure, utilizing thewell known polymerase chain reaction (PCR; the experimental embodimentset forth in Mullis, K. B., 1987, U.S. Pat. No. 4,683,202) which allowsfor the identification of sequences derived from genes which aredifferentially expressed. First, isolated RNA is reverse-transcribedinto single-stranded cDNA, utilizing standard techniques which are wellknown to those of skill in the art. Primers for the reversetranscriptase reaction may include, but are not limited to, oligodT-containing primers, preferably of the 3' primer type ofoligonucleotide described below.

Next, this technique uses pairs of PCR primers, as described below,which allow for the amplification of clones representing a random subsetof the RNA transcripts present within any given cell. Utilizingdifferent pairs of primers allows each of the mRNA transcripts presentin a cell to be amplified. Among such amplified transcripts may beidentified those which have been produced from differentially expressedgenes.

The 3' oligonucleotide primer of the primer pairs may contain an oligodT stretch of 10-13, preferably 11, dT nucleotides at its 5' end, whichhybridizes to the poly(A) tail of mRNA or to the complement of a cDNAreverse transcribed from an mRNA poly(A) tail. Second, in order toincrease the specificity of the 3' primer, the primer may contain one ormore, preferably two, additional nucleotides at its 3' end. Because,statistically, only a subset of the mRNA derived sequences present inthe sample of interest will hybridize to such primers, the additionalnucleotides allow the primers to amplify only a subset of the mRNAderived sequences present in the sample of interest. This is preferredin that it allows more accurate and complete visualization andcharacterization of each of the bands representing amplified sequences.

The 5' primer may contain a nucleotide sequence expected, statistically,to have the ability to hybridize to cDNA sequences derived from thetissues of interest. The nucleotide sequence may be an arbitrary one,and the length of the 5' oligonucleotide primer may range from about 9to about 15 nucleotides, with about 13 nucleotides being preferred.

Arbitrary primer sequences cause the lengths of the amplified partialcDNAs produced to be variable, thus allowing different clones to beseparated by using standard denaturing sequencing gel electrophoresis.

PCR reaction conditions should be chosen which optimize amplifiedproduct yield and specificity, and, additionally, produce amplifiedproducts of lengths which may be resolved utilizing standard gelelectrophoresis techniques. Such reaction conditions are well known tothose of skill in the art, and important reaction parameters include,for example, length and nucleotide sequence of oligonucleotide primersas discussed above, and annealing and elongation step temperatures andreaction times.

The pattern of clones resulting from the reverse transcription andamplification of the mRNA of two different cell types is displayed viasequencing gel electrophoresis and compared. Differentially expressedgenes are indicated by differences in the two banding patterns.

Once potentially differentially expressed gene sequences have beenidentified via bulk techniques such as, for example, those describedabove, the differential expression of such putatively differentiallyexpressed genes should be corroborated. Corroboration may beaccomplished via, for example, such well known techniques as Northernanalysis, quantitative RT PCR or RNase protection.

Upon corroboration, the differentially expressed genes may be furthercharacterized, and may be identified as target and/or fingerprint genes,as discussed, below, in Section 5.1.3.

Also, amplified sequences of differentially expressed genes obtainedthrough, for example, differential display may be used to isolate fulllength clones of the corresponding gene. The full length coding portionof the gene may readily be isolated, without undue experimentation, bymolecular biological techniques well known in the art. For example, theisolated differentially expressed amplified fragment may be labeled andused to screen a cDNA library. Alternatively, the labeled fragment maybe used to screen a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. As described, above, in this Section, the isolated, amplifiedgene fragments obtained through differential display have 5' terminalends at some random point within the gene and usually have 3' terminalends at a position corresponding to the 3' end of the transcribedportion of the gene. Once nucleotide sequence information from anamplified fragment is obtained, the remainder of the gene (i.e., the 5'end of the gene, when utilizing differential display) may be obtainedusing, for example, RT-PCR.

In one embodiment of such a procedure for the identification and cloningof full length gene sequences, RNA may be isolated, following standardprocedures, from an appropriate tissue or cellular source. A reversetranscription reaction may then be performed on the RNA using anoligonucleotide primer complimentary to the mRNA that corresponds to theamplified fragment, for the priming of first strand synthesis. Becausethe primer is anti-parallel to the mRNA, extension will proceed towardthe 5' end of the mRNA. The resulting RNA/DNA hybrid may then be"tailed" with guanines using a standard terminal transferase reaction,the hybrid may be digested with RNAase H, and second strand synthesismay then be primed with a poly-C primer. Using the two primers, the 5'portion of the gene is amplified using PCR. Sequences obtained may thenbe isolated and recombined with previously isolated sequences togenerate a full-length cDNA of the differentially expressed genes of theinvention. For a review of cloning strategies and recombinant DNAtechniques, see e.g., Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al.,1989, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y.

5.1.2. Methods for the Identification of Pathway Genes

Methods are described herein for the identification of pathway genes."Pathway gene", as used herein, refers to a gene whose gene productexhibits the ability to interact with gene products involved in bodyweight disorders and/or to interact with gene products which arerelevant to appetite or body weight regulation. A pathway gene may bedifferentially expressed and, therefore, may have the characteristics ofa target and/or fingerprint gene.

Any method suitable for detecting protein-protein interactions may beemployed for identifying pathway gene products by identifyinginteractions between gene products and gene products known to beinvolved in body weight disorders and/or involved in appetite or bodyregulation. Such known gene products may be cellular or extracellularproteins. Those gene products which interact with such known geneproducts represent pathway gene products and the genes which encode themrepresent pathway genes.

Among the traditional methods which may be employed areco-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns. Utilizing procedures such as theseallows for the identification of pathway gene products. Once identified,a pathway gene product may be used, in conjunction with standardtechniques, to identify its corresponding pathway gene. For example, atleast a portion of the amino acid sequence of the pathway gene productmay be ascertained using techniques well known to those of skill in theart, such as via the Edman degradation technique (see, e.g., Creighton,1983, "Proteins: Structures and Molecular Principles", W.H. Freeman &Co., N.Y., pp.34-49). The amino acid sequence obtained may be used as aguide for the generation of oligonucleotide mixtures that can be used toscreen for pathway gene sequences. Screening made be accomplished, forexample, by standard hybridization or PCR techniques. Techniques for thegeneration of oligonucleotide mixtures and the screening are well-known.(See, e.g., Ausubel, supra., and PCR Protocols: A Guide to Methods andApplications, 1990, Innis, M. et al., eds. Academic Press, Inc., NewYork).

Additionally, methods may be employed which result in the simultaneousidentification of pathway genes which encode the protein interactingwith a protein involved in body weight disorder states and/or appetiteand body weight regulation. These methods include, for example, probingexpression libraries with labeled protein known or suggested to beinvolved in body weight disorders and/or appetite or body weightregulation, using this protein in a manner similar to the well knowntechnique of antibody probing of λgt11 libraries.

One method which detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration only and not by way oflimitation. One version of this system has been described (Chien et al.,1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commerciallyavailable from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one consists of the DNA-binding domain of atranscription activator protein fused to a known protein, in this case,a protein known to be involved in body weight disorders and or processesrelevant to appetite and/or weight regulation, and the other consists ofthe transcription activator protein's activation domain fused to anunknown protein that is encoded by a cDNA which has been recombined intothis plasmid as part of a cDNA library. The plasmids are transformedinto a strain of the yeast Saccharomyces cerevisiae that contains areporter gene (e.g., latZ) whose regulatory region contains thetranscription activator's binding sites. Either hybrid protein alonecannot activate transcription of the reporter gene: the DNA-bindingdomain hybrid cannot because it does not provide activation function andthe activation domain hybrid cannot because it cannot localize to theactivator's binding sites. Interaction of the two hybrid proteinsreconstitutes the functional activator protein and results in expressionof the reporter gene, which is detected by an assay for the reportergene product.

The two-hybrid system or related methodology may be used to screenactivation domain libraries for proteins that interact with a known"bait" gene product. By way of example, and not by way of limitation,gene products known to be involved in body weight disorders and/orappetite or body weight regulation may be used as the bait geneproducts. These include but are not limited to the intracellular domainof receptors for such hormones as neuropeptide Y, galanin, interostatin,insulin, and CCK. Total genomic or cDNA sequences are fused to the DNAencoding an activation domain. This library and a plasmid encoding ahybrid of the bait gene product fused to the DNA-binding domain arecotransformed into a yeast reporter strain, and the resultingtransformants are screened for those that express the reporter gene. Forexample, and not by way of limitation, the bait gene can be cloned intoa vector such that it is translationally fused to the DNA encoding theDNA-binding domain of the GAL4 protein. These colonies are purified andthe library plasmids responsible for reporter gene expression areisolated. DNA sequencing is then used to identify the proteins encodedby the library plasmids.

A cDNA library of the cell line from which proteins that interact withbait gene product are to be detected can be made using methods routinelypracticed in the art. According to the particular system describedherein, for example, the cDNA fragments can be inserted into a vectorsuch that they are translationally fused to the activation domain ofGAL4. This library can be co-transformed along with the bait gene-GAL4fusion plasmid into a yeast strain which contains a lacZ gene driven bya promoter which contains GAL4 activation sequence. A cDNA encodedprotein, fused to GAL4 activation domain, that interacts with bait geneproduct will reconstitute an active GAL4 protein and thereby driveexpression of the lacZ gene. Colonies which express lacZ can be detectedby their blue color in the presence of X-gal. The cDNA can then bepurified from these strains, and used to produce and isolate the baitgene-interacting protein using techniques routinely practiced in theart.

Once a pathway gene has been identified and isolated, it may be furthercharacterized as, for example, discussed below, in Section 5.1.3.

5.1.3. Characterization of Differentially Expressed and Pathway Genes

Differentially expressed genes, such as those identified via the methodsdiscussed, above, in Section 5.1.1, and pathway genes, such as thoseidentified via the methods discussed, above, in Section 5.1.2, above, aswell as genes identified by alternative means, may be furthercharacterized by utilizing, for example, methods such as those discussedherein. Such genes will be referred to herein as "identified genes".

Analyses such as those described herein, yield information regarding thebiological function of the identified genes. An assessment of thebiological function of the differentially expressed genes, in addition,will allow for their designation as target and/or fingerprint genes.

Specifically, any of the differentially expressed genes whose furthercharacterization indicates that a modulation of the gene's expression ora modulation of the gene product's activity may ameliorate any of thebody weight disorders of interest will be designated "target genes", asdefined, above, in Section 5.1. Such target genes and target geneproducts, along with those discussed below, will constitute the focus ofthe compound discovery strategies discussed, below, in Section 5.3.Further, such target genes, target gene products and/or modulatingcompounds can be used as part of the body weight disorder treatmentmethods described, below, in Section 5.4.

Any of the differentially expressed genes whose further characterizationindicates that such modulations may not positively affect body weightdisorders of interest, but whose expression pattern contributes to agene expression "fingerprint" pattern correlative of, for example, abody weight disorder state will be designated a "fingerprint gene"."Fingerprint patterns" will be more fully discussed, below, in Section5.7.1. It should be noted that each of the target genes may alsofunction as fingerprint genes,as well as may all or a portion of thepathway genes.

It should further be noted that the pathway genes may also becharacterized according to techniques such as those described herein.Those pathway genes which yield information indicating that they aredifferentially expressed and that modulation of the gene's expression ora modulation of the gene product's activity may ameliorate any of thebody weight disorders of interest will be also be designated "targetgenes". Such target genes and target gene products, along with thosediscussed above, will constitute the focus of the compound discoverystrategies discussed, below, in Section 5.3 and can be used as part ofthe treatment methods described in Section 5.4, below.

It should be additionally noted that the characterization of one or moreof the pathway genes may reveal a lack of differential expression, butevidence that modulation of the gene's activity or expression may,nonetheless, ameliorate body weight disorder symptoms. In such cases,these genes and gene products would also be considered a focus of thecompound discovery strategies of Section 5.3, below.

In instances wherein a pathway gene's characterization indicates thatmodulation of gene expression or gene product activity may notpositively affect body weight disorders of interest, but whoseexpression is differentially expressed and contributes to a geneexpression fingerprint pattern correlative of, for example, a bodyweight disorder state, such pathway genes may additionally be designatedas fingerprint genes.

A variety of techniques can be utilized to further characterize theidentified genes. First, the nucleotide sequence of the identifiedgenes, which may be obtained by utilizing standard techniques well knownto those of skill in the art, may, for example, be used to revealhomologies to one or more known sequence motifs which may yieldinformation regarding the biological function of the identified geneproduct.

Second, an analysis of the tissue and/or cell type distribution of themRNA produced by the identified genes may be conducted, utilizingstandard techniques well known to those of skill in the art. Suchtechniques may include, for example, Northern, RNase protection andRT-PCR analyses. Such analyses provide information as to, for example,whether the identified genes are expressed in tissues or cell typesexpected to contribute to the body weight disorders of interest. Suchanalyses may also provide quantitative information regarding steadystate mRNA regulation, yielding data concerning which of the identifiedgenes exhibits a high level of regulation in, preferably, tissues whichmay be expected to contribute to the body weight disorders of interest.Additionally, standard in situ hybridization techniques may be utilizedto provide information regarding which cells within a given tissueexpress the identified gene. Such an analysis may provide informationregarding the biological function of an identified gene relative to agiven body weight disorder in instances wherein only a subset of thecells within the tissue is thought to be relevant to the body weightdisorder.

Third, the sequences of the identified genes may be used, utilizingstandard techniques, to place the genes onto genetic maps, e.g., mouse(Copeland, N. G. and Jenkins, N. A., 1991, Trends in Genetics 7:113-118)and human genetic maps (Cohen, D., et al., 1993, Nature 366:698-701).Such mapping information may yield information regarding the genes'importance to human disease by, for example, identifying genes which mapwithin genetic regions to which known genetic body weight disorders map.

Fourth, the biological function of the identified genes may be moredirectly assessed by utilizing relevant in vivo and in vitro systems. Invivo systems may include, but are not limited to, animal systems whichnaturally exhibit body weight disorder-like symptoms, or ones which havebeen engineered to exhibit such symptoms. Further, such systems mayinclude systems for the further characterization of body weightdisorders, and/or appetite or body weight regulation, and may include,but are not limited to, naturally occurring and transgenic animalsystems such as those described, above, in Section 5.1.1.1, and Section5.2.4.1, below. In vitro systems may include, but are not limited to,cell-based systems comprising cell types known or suspected ofcontributing to the body weight disorder of interest. Such cells may bewild type cells, or may be non-wild type cells containing modificationsknown to, or suspected of, contributing to the body weight disorder ofinterest. Such systems are discussed in detail, below, in Section5.2.4.2.

In further characterizing the biological function of the identifiedgenes, the expression of these genes may be modulated within the in vivoand/or in vitro systems, i.e., either overexpressed or underexpressedin, for example, transgenic animals and/or cell lines, and itssubsequent effect on the system then assayed. Alternatively, theactivity of the product of the identified gene may be modulated byeither increasing or decreasing the level of activity in the in vivoand/or in vitro system of interest, and its subsequent effect thenassayed.

The information obtained through such characterizations may suggestrelevant methods for the treatment of body weight disorders involvingthe gene of interest. Further, relevant methods for the control ofappetite and body weight regulation involving the gene of interest maybe suggested by information obtained from such characterizations. Forexample, treatment may include a modulation of gene expression and/orgene product activity. Characterization procedures such as thosedescribed herein may indicate where such modulation should involve anincrease or a decrease in the expression or activity of the gene or geneproduct of interest. Such methods of treatment are discussed, below, inSection 5.4.

5.2. Differentially Expressed and Pathway Genes

Identified genes, which include, but are not limited to, differentiallyexpressed genes such as those identified in Section 5.1.1, above, andpathway genes, such as those identified in Section 5.1.2, above, aredescribed herein. Specifically, the nucleic acid sequences and geneproducts of such identified genes are described. Further, antibodiesdirected against the identified genes' products, and cell- andanimal-based models by which the identified genes may be furthercharacterized and utilized are also discussed in this Section.

5.2.1. Differentially Expressed Gene Sequences

Differentially expressed nucleotide sequences are shown in FIGS. 3A,4-14, 16-17 and 20-21. Table 1 lists differentially expressed genes (P3,P13, F5, F49, murine C5, human C5, L31/F74, L7/L21, L29, L38, L37, L57,H27, F84 and L34) identified through, for example, the paradigmsdiscussed, above, in Section 5.1.1.1, and, below, in the examplespresented in Sections 6-10. Table 1 also summarizes informationregarding the further characterization of such genes. Table 2 lists E.coli clones, deposited with the Agricultural Research Service CultureCollection (NRRL), which contain sequences found within the F49 andhuman C5 genes listed in Table 1.

In Table 1, the differential expression patterns revealed via, forexample, one or more of the paradigm conditions described in Section5.1.1.1, above, are summarized under the column headed "ParadigmExpression Pattern". For each of the tested genes, the paradigm whichwas used and the difference in the expression of the gene inexperimental versus control tissues is shown. "" indicates that geneexpression is increased (i.e., there is an increase in the amount ofdetectable mRNA produced by a given gene) in experimental versus controltissue or cell type, while ".arrow-down dbl." indicates that geneexpression is decreased (i.e., there is an decrease in the amount ofdetectable mRNA produced by a given gene) in experimental versus controltissue or cell type. Further, "+" indicates that gene expression isactivated in experimental versus control tissue or cell type, i.e., mRNAis detectable in experimental tissue or cell type whereas none isdetectable in control tissue or cell type, while "-" would indicate thatgene expression is inactivated in experimental versus control tissue orcell type, i.e., while mRNA is detectable in control tissue or celltype, it is no longer detectable in experimental tissue or cell type."Detectable" as used herein, refers to levels of mRNA which aredetectable via standard differential display, Northern and/or RT-PCRtechniques which are well known to those of skill in the art."Increased" and "decreased", as used herein, refer to an increase ordecrease, respectively in level of mRNA present in experimental versuscontrol tissue or cell type which is detectable via standarddifferential display, Northern, and/or RT-PCT techniques which are wellknown to those of skill in the art.

Tissue expression patterns are also summarized in Table 1. The columnheaded "First Detection" indicates the first tissue or cell type inwhich differential expression of the gene was detected. The columnheaded "Tissue/Cell Dist." lists tissues and/or cell types in whichexpression of the gene has been tested and whether expression of thegene within a given tissue or cell type has been observed. Specifically,"+" indicates detectable mRNA from the gene of interest, while "-"refers to no detectable mRNA from the gene of interest. Unless otherwisenoted, "+" and "-" refer to both control and experimental samples."Detectable", as used herein, is as defined earlier in this Section.

Additionally, the physical locus to which the gene maps on the humanand/or mouse chromosome map is indicated in the column headed "Locus".Further, in instances wherein the genes correspond to genes known to befound in nucleic acid databases, references (i.e., citations and/or genenames) to such known genes are listed in the column headed "Ref".

The genes listed in Table 1 can be obtained using cloning methods wellknown to those of skill in the art, and which include, but are notlimited to, the use of appropriate probes to detect the genes within anappropriate cDNA or gDNA (genomic DNA) library. (See, for example,Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Hanson Laboratories, which is incorporated herein by reference inits entirety.) Probes for the sequences reported herein can be obtaineddirectly from the isolated clones deposited with the NRRL, as indicatedin Table 2, below. Alternatively, oligonucleotide probes for the genescan be synthesized based on the DNA sequences disclosed herein in FIGS.3A, 4-14, 16A, 16B 17A, 17B and 20-22. With respect to the previouslyreported genes, oligonucleotides can be synthesized or produced based onthe sequences provided for the previously known genes described in thefollowing references: glutamine synthetase (P3): Bhandari et al., 1991,J. Biol. Chem. 266:7784-7792; islet regenerating protein (P13): Unno, M.et al., 1987, J. Biol. Chem. 268:15974-15982; Terazono, K. et al., 1988,J. Biol. Chem. 263:2111-2114; Watanabe, T., 1990, J. Biol. Chem.265:7432-7439; alpha amylase (F5): Schibler, U. et al., 1986, in "OxfordSurveys on Eukaryotic Genes", Maclean, N., ed. 3:210, Oxford Univ.Press, New York; Schibler et al., 1982, J. Mol. Biol. 155:247-266; mousemajor urinary protein II (L31/F74): Shahan, K. et al., 1987, Mol. Cell.Biol. 1:1938-1946; mouse cytochrome C oxidase Subunit I (L7/L21): Bibbet al., 1981, Cell 26:167-180; mouse testosterone 15-alpha hydroxylase(L15): Squires, E. J. and Negism:, M., 1988, J. Biol. Chem.263:4166-4171; mouse 24p3 (L38): Flower, D. R. et al., 1991, Biochem.Biophys. Res. Comm. 180:69-74; Hraba-Renevey, S. et al., 1989, Oncogene4:601-608; mouse p6-5 (L37): Yamasaki, N. et al., 1987, Eur. J. Immunol.17:247-253; mouse orphan Nuclear hormone Reception (L57): Forman et al.,1994, Mol. Endocrinol. 81253-1261; autoantigen La (H27): GenbankAccession No. L00993; and mouse cytochrome p450 IID (F84): Matsunaga, E.et al., 1990, J. Mol. Evol. 30:155-169; mouse L34 (homolog of the humanlrp 130 gene): Hou, J. et al., 1994, In Vitro Cell. Dev. Biol. Anim.30A:111-114.

The probes can be used to screen cDNA libraries prepared from anappropriate cell, cell line or tissue in which the gene is transcribed.Appropriate cell lines can include, for example, preadipocyte cell linessuch as 3T3-A1 and TA1 mouse preadipocyte cell lines, liver cell lines,such as the Hepa-1-6 mouse liver cell line and the HepG2 human livercell line.

                                      TABLE 1                                     __________________________________________________________________________            Paradigm                                                                      Expression                                                                            First   Tissue/                                               Gene    Pattern Detection                                                                             Cell Dist.                                                                            Locus                                                                             Ref                                       __________________________________________________________________________    P3 (SEQ. ID                                                                           ↑Pancreas                                                                       Pancreas            .sup.1                                    NO:4, 39)                                                                             (fasted)                                                              P13 (SEQ. ID                                                                          ↑Pancreas                                                                       Pancreas            .sup.2                                    NO:7, 40-42)                                                                          (fasted)                                                              F5 (SEQ. ID                                                                           ↑Adipose                                                                        Adipose             .sup.3                                    NO:12)  (fasted)                                                              F49 (SEQ. ID                                                                          +Adipose                                                                              Adipose Adipose Chrom                                         NO:34)  (db/db)          (+)    2                                                                     db/db;                                                                        (-) lean                                                                      control!                                                                      Muscle                                                                        (-)                                                                           Small                                                                         Intestine                                                                     (-)                                                                           Hypothalamus                                                                  (-)                                                                           Liver (-)                                                                     Pancreas                                                                      (-)                                                   murine C5.sup.10                                                                      ↑Adipose                                                                        Adipose                                                       (SEQ. ID                                                                              (ob/ob and                                                            NO: 36) db/db)                                                                L31/F74 ↑Liver and                                                                      Adipose;                                                                              Liver       .sup.4                                    (SEQ. ID                                                                              Adipose Liver   (+)                                                   NO:16)  (ob/ob and      Adipose                                                       db/db)          (+)                                                           ↑Liver    Muscle                                                        (underweight)   (-)                                                   L7/L21 (SEQ.                                                                          ↑Liver                                                                          Liver               .sup.5                                    ID NO:18)                                                                             (fasted,                                                                      ob/ob, and                                                                    db/db)                                                                L29 (SEQ. ID                                                                          ↑Liver                                                                          Liver               .sup.6                                    NO:20)  (ob/ob)                                                               L38 (SEQ. ID                                                                          ↑Liver (ob/ob                                                                   Liver               .sup.7                                    NO:22, 43)                                                                            and db/db)                                                            L37 (SEQ.                                                                             ↑Liver                                                                          Liver               .sup.8                                    NO:25, 44-                                                                            (ob/ob)                                                               45)                                                                           L57 (SEQ. ID                                                                          ↑Liver                                                                          Liver               .sup.9                                    NO:29, 46-                                                                            (underweight)                                                         48)                                                                           Human C5                Heart                                                 (SEQ ID                 (+)                                                   NO.:38)                 Brain (-)                                                                     Placenta                                                                      (+)                                                                           Lung (+)                                                                      Liver                                                                         (+)                                                                           Muscle                                                                        (+)                                                                           Kidney                                                                        (+)                                                                           Pancreas                                                                      (+)                                                   H27 (SEQ ID                                                                           ↑Hypothalamus                                                                   Hypothalamus        .sup.11                                   NO:49)  (fasted)                                                              F84 (SEQ ID                                                                           ↑Adipose                                                                        Adipose             .sup.12                                   NO:52, 54)                                                                            (underweight)                                                         L34 (SEQ ID                                                                           ↑Liver                                                                          Liver               .sup.13                                   NO:57)  (ob/ob)                                                               __________________________________________________________________________     .sup.1 Mouse glutamine synthetase:. Bhandari et al., 1991, J. Biol. Chem.     266:7784-7792.                                                                .sup.2 Mouse islet regenerating protein: Unno, M. et al., 1993, J. Biol.      Chem. 268:15974-15982.                                                        .sup.3 Mouse amylase: Schibler, U. et al., 1986, in "Oxford Surveys on        Eukaryotic Genes", Maclean, N., ed., 3:210, Oxford Univ. Press, New York;     Schibler et al., 1982, J. Mol. Biol. 155:247-266.                             .sup.4 Mouse major urinary protein II: Shahan, K. et al., 1987, Mol. Cell     Biol. 7:1938-1946.                                                            .sup.5 Mouse cytochrome C oxidase Subunit I: Raikhinstein, M. and             Hankoglu, I., 1993, Proc. Natl. Acad. Sci. USA.90:10509-10513; Bibb et        al., 1981, Cell 26:167-180.                                                   .sup.6 Mouse testosterone 15α hydroxylase: Squires, E. J. and           Negishi, M., 1988, J. Biol. Chem. 263:4166-4171.                              .sup.7 Mouse 24p3: Flower, D. R. et al., 1991, Biochem. Biophys. Res.         Comm. 180:69-74; HrabaRenevey, S. et al., 1989; Oncogene 4:601-608.           .sup.8 Mouse p65: Yamasaki, N. et al., 1987, Eur. J. Immunol. 17:247-253.     .sup.9 Mouse orphan nuclear hormone receptor: Forman et al., 1994, Mol.       Endocrinol. 8:1253-1261.                                                      .sup.10 The mouse C5 sequence was first identified via sequence homology.     C5 was then subsequently tested in ob and db mice, at which time it was       identified to represent a differentially expressed gene sequence.             .sup.11 Mouse autoantigen La: Genbank Accesssion No. L00993.                  .sup.12 Mouse cytochrome p450 IID: Matsunaga, E. et al., 1990, J. Mol.        Evol. 30:155-169.                                                             .sup.13 The mouse L34 gene represents the mouse homolog of the human 1rp      130 gene: Hou, J. et al., 1994, In Vitro Cell. Dev. Biol. Anim.               30A:111-114.                                                             

Table 2, below, lists isolated cDNA clones that contain genes listed inTable 1.

                  TABLE 2                                                         ______________________________________                                        GENE                cDNA CLONE                                                ______________________________________                                        F49                 famf049a                                                  human C5            fahs005a                                                  ______________________________________                                    

As used herein, "differentially expressed gene" (i.e. target andfingerprint gene) or "pathway gene" refers to (a) a gene containing: atleast one of the DNA sequences disclosed herein (as shown in FIGS. 3A,4-14, 16A, 16B, 17A, 17B, and 20-22), or contained in the clones listedin Table 2, as deposited with the NRRL; (b) any DNA sequence thatencodes the amino acid sequence encoded by: the DNA sequences disclosedherein (as shown in FIGS. 3A, 4-14, 16A, 16B, 17A, 17B, and 20-22),contained in the clones listed in Table 2, as deposited with the NRRL,or contained within the coding region of the gene to which the DNAsequences disclosed herein (as shown in FIGS. 3A, 4-14, 16A, 16B, 17A,17B, and 20-22) or contained in the clones listed in Table 2, asdeposited with the NRRL, belong; (c) any DNA sequence that hybridizes tothe complement of: the coding sequences disclosed herein (as shown inFIGS. 3A, 4-14, 16A, 16B, 17A, 17B, and 20-22), contained in cloneslisted in Table 2, as deposited with the NRRL, or contained within thecoding region of the gene to which the DNA sequences disclosed herein(as shown in FIGS. 3A, 4-14, 16A, 16B, 17A, 17B, and 20-22) or containedin the clones listed in Table 2, as deposited with the NRRL, belong,under highly stringent conditions, e.g., hybridization to filter-boundDNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° ,and washing in 0.1× SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds.,1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3),and encodes a gene product functionally equivalent to a gene productencoded by a gene of (a), above; and/or (d) any DNA sequence thathybridizes to the complement of: the coding sequences disclosed herein,(as shown in FIGS. 3A, 4-14, 16A, 16B, 17A, 17B, and 20-22) contained inthe clones listed in Table 2, as deposited with the NRRL, or containedwithin the coding region of the gene to which DNA sequences disclosedherein (as shown in FIGS. 3A, 4-14, 16A, 16B, 17A, 17B, and 20-22) orcontained in the clones, listed in Table 2, as deposited with the NRRL,belong, under less stringent conditions, such as moderately stringentconditions, e.g., washing in 0.2× SSC/0.1% SDS at 42° C. (Ausubel etal., 1989, supra), yet which still encodes a gene product, functionallyequivalent to a gene product encoded by a gene of (a), above.

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, theDNA sequences (a) through (d), in the preceding paragraph. Suchhybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides ("oligos"), highly stringentconditions may refer, e.g., to washing in 6× SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may act as target gene antisense molecules,useful, for example, in target gene regulation and/or as antisenseprimers in amplification reactions of target, fingerprint, and/orpathway gene nucleic acid sequences. Further, such sequences may be usedas part of ribozyme and/or triple helix sequences, also useful fortarget gene regulation. Still further, such molecules may be used ascomponents of diagnostic methods whereby the presence of, orpredisposition to, a body weight disorder, may be detected.

The invention also encompasses (a) DNA vectors that contain any of theforegoing coding sequences and/or their complements (i.e., antisense);(b) DNA expression vectors that contain any of the foregoing codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences; and (c) genetically engineeredhost cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Theinvention includes fragments of any of the DNA sequences disclosedherein.

In addition to the gene sequences described above, homologues of thesegene sequences as may, for example, be present in other species,preferably human in instances wherein the above-described gene sequencesare not human gene sequences, may be identified and isolated, withoutundue experimentation, by molecular biological techniques well known inthe art. Further, there may exist genes at other genetic loci within thegenome that encode proteins which have extensive homology to one or moredomains of such gene products. These genes may also be identified viasimilar techniques.

For example, an isolated differentially expressed gene sequence may belabeled and used to screen a cDNA library constructed from mRNA obtainedfrom the organism of interest. Hybridization conditions will be of alower stringency when the cDNA library was derived from an organismdifferent from the type of organism from which the labeled sequence wasderived. Alternatively, the labeled fragment may be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions. Such low stringency conditions willbe well known to those of skill in the art, and will vary predictablydepending on the specific organisms from which the library and thelabeled sequences are derived. For guidance regarding such conditionssee, for example, Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989,Current Protocols in Molecular Biology, (Green Publishing Associates andWiley Interscience, N.Y.).

Further, a previously unknown differentially expressed or pathwaygene-type sequence may be isolated by performing PCR using twodegenerate oligonucleotide primer pools designed on the basis of aminoacid sequences within the gene of interest. The template for thereaction may be cDNA obtained by reverse transcription of mRNA preparedfrom human or non-human cell lines or tissue known or suspected toexpress a differentially expressed or pathway gene allele. The PCRproduct may be subcloned and sequenced to insure that the amplifiedsequences represent the sequences of a differentially expressed orpathway gene-like nucleic acid sequence.

The PCR fragment may then be used to isolate a full length cDNA clone bya variety of methods. For example, the amplified fragment may be used toscreen a bacteriophage cDNA library. Alternatively, the labeled fragmentmay be used to screen a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source. A reversetranscription reaction may be performed on the RNA using anoligonucleotide primer specific for the most 5' end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be "tailed" with guanines using a standardterminal transferase reaction, the hybrid may be digested with RNAase H,and second strand synthesis may then be primed with a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment may easily beisolated. For a review of cloning strategies which may be used, seee.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, (Green Publishing Associates and WileyInterscience, N.Y.).

In cases where the differentially expressed or pathway gene identifiedis the normal, or wild type, gene, this gene may be used to isolatemutant alleles of the gene. Such an isolation is preferable in processesand disorders which are known or suspected to have a genetic basis.Mutant alleles may be isolated from individuals either known orsuspected to have a genotype which contributes to body weight disordersymptoms. Mutant alleles and mutant allele products may then be utilizedin the therapeutic and diagnostic assay systems described below.

A cDNA of the mutant gene may be isolated, for example, by using PCR, atechnique which is well known to those of skill in the art. In thiscase, the first cDNA strand may be synthesized by hybridizing a oligo-dToligonucleotide to mRNA isolated from tissue known to, or suspected of,being expressed in an individual putatively carrying the mutant allele,and by extending the new strand with reverse transcriptase. The secondstrand of the cDNA is then synthesized using an oligonucleotide thathybridizes specifically to the 5'- end of the normal gene. Using thesetwo primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant gene to that of the normal gene, the mutation(s)responsible for the loss or alteration of function of the mutant geneproduct can be ascertained.

Alternatively, a genomic or cDNA library can be constructed and screenedusing DNA or RNA, respectively, from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. The normal gene or any suitable fragmentthereof may then be labeled and used as a probe to identify thecorresponding mutant allele in the library. The clone containing thisgene may then be purified through methods routinely practiced in theart, and subjected to sequence analysis as described, above, in thisSection.

Additionally, an expression library can be constructed utilizing DNAisolated from or cDNA synthesized from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. In this manner, gene products made by theputatively mutant tissue may be expressed and screened using standardantibody screening techniques in conjunction with antibodies raisedagainst the normal gene product, as described, below, in Section 5.2.3.(For screening techniques, see, for example, Harlow, E. and Lane, eds.,1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, ColdSpring Harbor.) In cases where the mutation results in an expressed geneproduct with altered function (e.g., as a result of a missensemutation), a polyclonal set of antibodies are likely to cross-react withthe mutant gene product. Library clones detected via their reaction withsuch labeled antibodies can be purified and subjected to sequenceanalysis as described in this Section, above.

5.2.2. Differentially Expressed and Pathway Gene Products

Differentially expressed and pathway gene products include thoseproteins encoded by the differentially expressed and pathway genesequences described in Section 5.2.1, above, as for example, thepeptides listed in FIGS. 14 (SEQ ID NO: 34), 16A, 16B (SEQ ID NO: 36),17A, 17B (SEQ. ID NO.: 56) and 18 (SEQ ID NO: 51).

In addition, differentially expressed and pathway gene products mayinclude proteins that represent functionally equivalent gene products.Such an equivalent differentially expressed or pathway gene product maycontain deletions, additions or substitutions of amino acid residueswithin the amino acid sequence encoded by the differentially expressedor pathway gene sequences described, above, in Section 5.2.1, but whichresult in a silent change, thus producing a functionally equivalentdifferentially expressed or pathway gene product. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. "Functionallyequivalent", as utilized herein, refers to either a protein capable ofexhibiting a substantially similar in vivo activity as the endogenousdifferentially expressed or pathway gene products encoded by thedifferentially expressed or pathway gene sequences described in Section5.2.1, above. Alternatively, when utilized as part of assays such asthose described, below, in Section 5.3, "functionally equivalent" mayrefer to peptides capable of interacting with other cellular orextracellular molecules in a manner substantially similar to the way inwhich the corresponding portion of the endogenous differentiallyexpressed or pathway gene product would.

The differentially expressed or pathway gene products may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing the differentially expressed or pathway genepolypeptides and peptides of the invention by expressing nucleic acidencoding differentially expressed or pathway gene sequences aredescribed herein. Methods which are well known to those skilled in theart can be used to construct expression vectors containingdifferentially expressed or pathway gene protein coding sequences andappropriate transcriptional/translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. See, forexample, the techniques described in Sambrook et al., 1989, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. whichis incorporated by reference herein in their entirety, and Ausubel,1989, supra. Alternatively, RNA capable of encoding differentiallyexpressed or pathway gene protein sequences may be chemicallysynthesized using, for example, synthesizers. See, for example, thetechniques described in "Oligonucleotide Synthesis", 1984, Gait, M. J.ed., IRL Press, Oxford, which is incorporated by reference herein in itsentirety.

A variety of host-expression vector systems may be utilized to expressthe differentially expressed or pathway gene coding sequences of theinvention. Such host-expression systems represent vehicles by which thecoding sequences of interest may be produced and subsequently purified,but also represent cells which may, when transformed or transfected withthe appropriate nucleotide coding sequences, exhibit the differentiallyexpressed or pathway gene protein of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. Subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing differentiallyexpressed or pathway gene protein coding sequences; yeast (e.g.Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing the differentially expressed or pathway gene proteincoding sequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the differentiallyexpressed or pathway gene protein coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingdifferentially expressed or pathway gene protein coding sequences; ormammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for thedifferentially expressed or pathway gene protein being expressed. Forexample, when a large quantity of such a protein is to be produced, forthe generation of antibodies or to screen peptide libraries, forexample, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which thedifferentially expressed or pathway gene protein coding sequence may beligated individually into the vector in frame with the lacZ codingregion so that a fusion protein is produced; pIN vectors (Inouye &Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned Target gene protein can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The differentially expressed or pathwaygene coding sequence may be cloned individually into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).Successful insertion of differentially expressed or pathway gene codingsequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed. (E.g., see Smith et al., 1983, J. Viol.46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the differentially expressed or pathway gene coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing differentially expressedor pathway gene protein in infected hosts. (e.g., See Logan & Shenk,1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiationsignals may also be required for efficient translation of inserteddifferentially expressed or pathway gene coding sequences. These signalsinclude the ATG initiation codon and adjacent sequences. In cases wherean entire differentially expressed or pathway gene, including its owninitiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only a portion of thedifferentially expressed or pathway gene coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe differentially expressed or pathway gene protein may be engineered.Rather than using expression vectors which contain viral origins ofreplication, host cells can be transformed with DNA controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express the differentially expressed or pathway gene protein. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of thedifferentially expressed or pathway gene protein.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30:147) genes.

When used as a component in assay systems such as those describedherein, the differentially expressed or pathway gene protein may belabeled, either directly or indirectly, to facilitate detection of acomplex formed between the differentially expressed or pathway geneprotein and a test substance. Any of a variety of suitable labelingsystems may be used including but not limited to radioisotopes such as¹²⁵ I; enzyme labelling systems that generate a detectable colorimetricsignal or light when exposed to substrate; and fluorescent labels.

Where recombinant DNA technology is used to produce the differentiallyexpressed or pathway gene protein for such assay systems, it may beadvantageous to engineer fusion proteins that can facilitate labeling,immobilization and/or detection.

Fusion proteins which facilitate solubility can include, but are notlimited to soluble Ig-tailed fusion proteins. Methods for engineeringsuch soluble Ig-tailed fusion proteins are well known to those of skillin the art. See, for example, U.S. Pat. No. 5,116,964, which isincorporated herein by reference in its entirety.

Indirect labeling involves the use of a protein, such as a labeledantibody, which specifically binds to a differentially expressed orpathway gene product. Such antibodies include but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by an Fab expression library.

5.2.3. Antibodies Specific for Differentially Expressed or Pathway GeneProducts

Described herein are methods for the production of antibodies capable ofspecifically recognizing one or more differentially expressed or pathwaygene epitopes. Such antibodies may include, but are not limited topolyclonal antibodies, monoclonal antibodies (mAbs), humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂fragments, fragments produced by a FAb expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. Such antibodies may be used, for example, in thedetection of a fingerprint, target, or pathway gene in a biologicalsample, or, alternatively, as a method for the inhibition of abnormaltarget gene activity. Thus, such antibodies may be utilized as part ofbody weight disorder treatment methods, and/or may be used as part ofdiagnostic techniques whereby patients may be tested for abnormal levelsof fingerprint, target, or pathway gene proteins, or for the presence ofabnormal forms of the such proteins.

For the production of antibodies to a differentially expressed orpathway gene, various host animals may be immunized by injection with adifferentially expressed or pathway gene protein, or a portion thereof.Such host animals may include but are not limited to rabbits, mice, andrats, to name but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection withdifferentially expressed or pathway gene product supplemented withadjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of "chimericantibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to producedifferentially expressed or pathway gene-single chain antibodies. Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab')₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

5.2.4. Cell- and Animal-based Model Systems

Described herein are cell- and animal-based systems which act as modelsfor body weight disorders. These systems may be used in a variety ofapplications. For example, the animal-based model systems can beutilized to identify differentially expressed genes via one of theparadigms described, above, in Section 5.1.1.1. Cell- and animal-basedmodel systems may be used to further characterize differentiallyexpressed and pathway genes, as described, above, in Section 5.1.3. Suchfurther characterization may, for example, indicate that adifferentially expressed gene is a target gene. Second, such assays maybe utilized as part of screening strategies designed to identifycompounds which are capable of ameliorating body weight disordersymptoms, as described, below. Thus, the animal- and cell-based modelsmay be used to identify drugs, pharmaceuticals, therapies andinterventions which may be effective in treating such body weightdisorders. In addition, as described in detail, below, in Section 5.6,such animal models may be used to determine the LD₅₀ and the ED₅₀ inanimal subjects, and such data can be used to determine the in vivoefficacy of potential body weight disorder treatments.

5.2.4.1. Animal-Based Systems

Animal-based model systems of body weight disorders may include, but arenot limited to, non-recombinant and engineered transgenic animals.

Non-recombinant animal models for body weight disorders may include, forexample, genetic models. Such genetic body disorder models may include,for example, mouse models of obesity such as mice homozygous for theautosomal recessive ob, db, or tub alleles.

Non-recombinant, non-genetic animal models of body weight disorders mayinclude, for example, rat models in which bilateral lesions exist in theventromedial hypothalamus, leading to hyperphagia and gross obesity, orin which ventrolateral hypothalamus lesions exist, which lead toaphagia. Further, mice which, as newborns, are fed mono-sodium-glutamate(MSG) develop obesity, and may, therefore, also be utilized as animalmodels for body weight disorders.

Additionally, animal models exhibiting body weight disorder-likesymptoms may be engineered by utilizing, for example, target genesequences such as those described, above, in Section 5.2, in conjunctionwith techniques for producing transgenic animals that are well known tothose of skill in the art. For example, target gene sequences may beintroduced into, and overexpressed in, the genome of the animal ofinterest, or, if endogenous target gene sequences are present, they may,either be overexpressed or, alternatively, may be disrupted in order tounderexpress or inactivate target gene expression.

In order to overexpress a target gene sequence, the coding portion ofthe target gene sequence may be ligated to a regulatory sequence whichis capable of driving gene expression in the animal and cell type ofinterest. Such regulatory regions will be well known to those of skillin the art, and may be utilized in the absence of undue experimentation.

For underexpression of an endogenous target gene sequence, such asequence may be isolated and engineered such that when reintroduced intothe genome of the animal of interest, the endogenous target gene alleleswill be inactivated. Preferably, the engineered target gene sequence isintroduced via gene targeting such that the endogenous target sequenceis disrupted upon integration of the engineered target gene sequenceinto the animal's genome. Gene targeting is discussed, below, in thisSection.

Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,e.g., baboons, squirrels, monkeys, and chimpanzees may be used togenerate body weight disorder animal models.

Any technique known in the art may be used to introduce a target genetransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. (See,for example, techniques described by Jakobovits, 1994, Curr. Biol.4:761-763). The transgene may be integrated as a single transgene or inconcatamers, e.g., head-to-head tandems or head-to-tail tandems. Thetransgene may also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lasko etal. (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236).The regulatory sequences required for such a cell-type specificactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art.

When it is desired that the target gene transgene be integrated into thechromosomal site of the endogenous target gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous targetgene of interest are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of, the nucleotide sequence of the endogenous target gene.The transgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous gene of interest in only thatcell type, by following, for example, the teaching of Gu et al. (Gu, H.et al., 1994, Science 265:103-106). The regulatory sequences requiredfor such a cell-type specific inactivation will depend upon theparticular cell type of interest, and will be apparent to those of skillin the art.

Once transgenic animals have been generated, the expression of therecombinant target gene and protein may be assayed utilizing standardtechniques. Initial screening may be accomplished by Southern blotanalysis or PCR techniques to analyze animal tissues to assay whetherintegration of the transgene has taken place. The level of mRNAexpression of the transgene in the tissues of the transgenic animals mayalso be assessed using techniques which include but are not limited toNorthern blot analysis of tissue samples obtained from the animal, insitu hybridization analysis, and RT-PCR. Samples of targetgene-expressing tissue, may also be evaluated immunocytochemically usingantibodies specific for the target gene transgene gene product ofinterest.

The target gene transgenic animals that express target gene mRNA ortarget gene transgene peptide (detected immunocytochemically, usingantibodies directed against target gene product epitopes) at easilydetectable levels should then be further evaluated to identify thoseanimals which display characteristic body weight disorder-like symptoms.Such symptoms may include, for example, obesity, anorexia, and anabnormal food intake. Additionally, specific cell types within thetransgenic animals may be analyzed and assayed for cellular phenotypescharacteristic of body weight disorders. Such cellular phenotypes mayinclude, for example, abnormal adipocyte differentiation (e.g., abnormalpreadipocyte/adipoctye differentiation) and metabolism. Further, suchcellular phenotypes may include as assessment of a particular cell typesfingerprint pattern of expression and its comparison to knownfingerprint expression profiles of the particular cell type in animalsexhibiting body weight disorders. Such transgenic animals serve assuitable model systems for body weight disorders.

Once target gene transgenic founder animals are produced (i.e., thoseanimals which express target gene proteins in cells or tissues ofinterest, and which, preferably, exhibit symptoms of body weightdisorders), they may be bred, inbred, outbred, or crossbred to producecolonies of the particular animal. Examples of such breeding strategiesinclude but are not limited to: outbreeding of founder animals with morethan one integration site in order to establish separate lines;inbreeding of separate lines in order to produce compound target genetransgenics that express the target gene transgene of interest at higherlevels because of the effects of additive expression of each target genetransgene; crossing of heterozygous transgenic animals to produceanimals homozygous for a given integration site in order to both augmentexpression and eliminate the possible need for screening of animals byDNA analysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; breeding animals to different inbredgenetic backgrounds so as to examine effects of modifying alleles onexpression of the target gene transgene and the development of bodyweight disorder-like symptoms. One such approach is to cross the targetgene transgenic founder animals with a wild type strain to produce an F1generation that exhibits body weight disorder-like symptoms, such asobesity, anorexia, and abnormal food intake. The F1 generation may thenbe inbred in order to develop a homozygous line, if it is found thathomozygous target gene transgenic animals are viable.

5.2.4.2. Cell-Based Assays

Cells that contain and express target gene sequences which encode targetgene protein, and, further, exhibit cellular phenotypes associated witha body weight disorder of interest, may be utilized to identifycompounds that exhibit an ability to ameliorate body weight disordersymptoms. Cellular phenotypes which may indicate an ability toameliorate body weight disorders may include, for example, inhibition ofadipose cell differentiation (e.g., an inhibition of differentiation ofpreadipocytes into adipocytes) and an inhibition of the ability ofadipocytes to synthesize fat.

Further, the fingerprint pattern of gene expression of cells of interestmay be analyzed and compared to the normal, non-body weight disorderfingerprint pattern. Those compounds which cause cells exhibiting bodyweight disorder-like cellular phenotypes to produce a fingerprintpattern more closely resembling a normal fingerprint pattern for thecell of interest may be considered candidates for further testingregarding an ability to ameliorate body weight disorder symptoms.

Cells which be utilized for such assays may, for example, includenon-recombinant cell lines, such as preadipocyte cell lines such as3T3-L1 and TA1 mouse preadipocyte cell lines, liver cell lines, such asthe Hepa1-6 mouse liver cell line, and the HepG2 human liver cell line.

Further, cells which may be used for such assays may also includerecombinant, transgenic cell lines. For example, the body weightdisorder animal models of the invention, discussed, above, in Section5.2.4.1, may be used to generate cell lines, containing one or more celltypes involved in body weight disorders, that can be used as cellculture models for this disorder. While primary cultures derived fromthe body weight disorder transgenic animals of the invention may beutilized, the generation of continuous cell lines is preferred. Forexamples of techniques which may be used to derive a continuous cellline from the transgenic animals, see Small et al., 1985, Mol. CellBiol. 5:642-648.

Alternatively, cells of a cell type known to be involved in body weightdisorders may be transfected with sequences capable of increasing ordecreasing the amount of target gene expression within the cell. Forexample, target gene sequences may be introduced into, and overexpressedin, the genome of the cell of interest, or, if endogenous target genesequences are present, they may either be overexpressed or,alternatively, be disrupted in order to underexpress or inactivatetarget gene expression.

In order to overexpress a target gene sequence, the coding portion ofthe target gene sequence may be ligated to a regulatory sequence whichis capable of driving gene expression in the cell type of interest. Suchregulatory regions will be well known to those of skill in the art, andmay be utilized in the absence of undue experimentation.

For underexpression of an endogenous target gene sequence, such asequence may be isolated and engineered such that when reintroduced intothe genome of the cell type of interest, the endogenous target genealleles will be inactivated. Preferably, the engineered target genesequence is introduced via gene targeting such that the endogenoustarget sequence is disrupted upon integration of the engineered targetgene sequence into the cell's genome. Gene targeting is discussed,above, in Section 5.4.2.1.

Transfection of target gene sequence nucleic acid may be accomplished byutilizing standard techniques. See, for example, Ausubel, 1989, supra.Transfected cells should be evaluated for the presence of therecombinant target gene sequences, for expression and accumulation oftarget gene mRNA, and for the presence of recombinant target geneprotein production. In instances wherein a decrease in target geneexpression is desired, standard techniques may be used to demonstratewhether a decrease in endogenous target gene expression and/or in targetgene product production is achieved.

5.3. Screening Assays for Compounds that Interact with the Target GeneProduct

The following assays are designed to identify compounds that bind totarget gene products, bind to other cellular proteins that interact witha target gene product, and to compounds that interfere with theinteraction of the target gene product with other cellular proteins.Such compounds may include, but are not limited to, other cellularproteins. Methods for the identification of such cellular proteins aredescribed, below, in Section 5.3.2.

Compounds may include, but are not limited to, peptides such as, forexample, soluble peptides, including but not limited to, Ig-tailedfusion peptides, comprising extracellular portions of target geneproduct transmembrane receptors, and members of random peptidelibraries; (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84;Houghten, R. et al., 1991, Nature 354:84-86), made of D- and/orL-configuration amino acids, phosphopeptides (including, but not limitedto members of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab')₂ and FAb expression library fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of thetarget gene product, and for ameliorating body weight disorders. Ininstances, for example, whereby a body weight disorder situation resultsfrom a lower overall level of target gene expression, target geneproduct, and/or target gene product activity in a cell or tissueinvolved in such a body weight disorder, compounds that interact withthe target gene product may include ones which accentuate or amplify theactivity of the bound target gene protein. Such compounds would bringabout an effective increase in the level of target gene activity, thusameliorating symptoms. In instances whereby mutations within the targetgene cause aberrant target gene proteins to be made which have adeleterious effect that leads to a body weight disorder, compounds thatbind target gene protein may be identified that inhibit the activity ofthe bound target gene protein. Assays for testing the effectiveness ofcompounds, identified by, for example, techniques such as thosedescribed in Section 5.3.1-5.3.3, are discussed, below, in Section5.3.4.

5.3.1. In Vitro Screening Assays for Compounds that Bind to the TargetGene Product

In vitro systems may be designed to identify compounds capable ofbinding the target gene products of the invention. Compounds identifiedmay be useful, for example, in modulating the activity of wild typeand/or mutant target gene products, may be useful in elaborating thebiological function of the target gene product, may be utilized inscreens for identifying compounds that disrupt normal target geneproduct interactions, or may in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to thetarget gene product involves preparing a reaction mixture of the targetgene product and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoringtarget gene product or the test substance onto a solid phase anddetecting target gene product/test compound complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, the target gene product may be anchored onto a solid surface,and the test compound, which is not anchored, may be labeled, eitherdirectly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for target geneproduct or the test compound to anchor any complexes formed in solution,and a labeled antibody specific for the other component of the possiblecomplex to detect anchored complexes.

5.3.2. Assays for Cellular Proteins that Interact with the Target GeneProtein

Any method suitable for detecting protein-protein interactions may beemployed for identifying novel target protein-cellular or extracellularprotein interactions. These methods are outlined in section 5.1.2.,above, for the identification of pathway genes, and may be utilizedherein with respect to the identification of proteins which interactwith identified target proteins.

5.3.3. Assays for Compounds that Interfere with Target GeneProduct/Cellular Macromolecule Interaction

The target gene products of the invention may, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins.Such macromolecules may include, but are not limited to, nucleic acidmolecules and those proteins identified via methods such as thosedescribed, above, in Section 5.3.2. For purposes of this discussion,such cellular and extracellular macromolecules are referred to herein as"binding partners". Compounds that disrupt such interactions may beuseful in regulating the activity of the target gene product, especiallymutant target gene products. Such compounds may include, but are notlimited to molecules such as antibodies, peptides, and the like, asdescribed, for example, in Section 5.3.1. above.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the target gene product and itscellular or extracellular binding partner or partners involves preparinga reaction mixture containing the target gene product, and the bindingpartner under conditions and for a time sufficient to allow the two tointeract and bind, thus forming a complex. In order to test a compoundfor inhibitory activity, the reaction mixture is prepared in thepresence and absence of the test compound. The test compound may beinitially included in the reaction mixture, or may be added at a timesubsequent to the addition of target gene product and its cellular orextracellular binding partner. Control reaction mixtures are incubatedwithout the test compound or with a placebo. The formation of anycomplexes between the target gene protein and the cellular orextracellular binding partner is then detected. The formation of acomplex in the control reaction, but not in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of the target gene protein and the interactivebinding partner. Additionally, complex formation within reactionmixtures containing the test compound and normal target gene protein mayalso be compared to complex formation within reaction mixturescontaining the test compound and a mutant target gene protein. Thiscomparison may be important in those cases wherein it is desirable toidentify compounds that disrupt interactions of mutant but not normaltarget gene proteins.

The assay for compounds that interfere with the interaction of thetarget gene products and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the target gene product or the binding partner onto asolid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the target gene products and the bindingpartners, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with thetarget gene protein and interactive cellular or extracellular bindingpartner. Alternatively, test compounds that disrupt preformed complexes,e.g. compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are described briefly below.

In a heterogeneous assay system, either the target gene protein or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface, while the non-anchored species is labeled, eitherdirectly or indirectly. In practice, microtiter plates are convenientlyutilized. The anchored species may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished simplyby coating the solid surface with a solution of the target gene productor binding partner and drying. Alternatively, an immobilized antibodyspecific for the species to be anchored may be used to anchor thespecies to the solid surface. The surfaces may be prepared in advanceand stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, may bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the target gene proteinand the interactive cellular or extracellular binding partner isprepared in which either the target gene product or its binding partnersis labeled, but the signal generated by the label is quenched due tocomplex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubensteinwhich utilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances which disrupt target geneprotein/cellular or extracellular binding partner interaction can beidentified.

In a particular embodiment, the target gene product can be prepared forimmobilization using recombinant DNA techniques described in Section5.2.1, above. For example, the target gene coding region can be fused toa glutathione-S-transferase (GST) gene using a fusion vector, such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion protein. The interactive cellular or extracellularbinding partner can be purified and used to raise a monoclonal antibody,using methods routinely practiced in the art and described above, inSection 5.2.3. This antibody can be labeled with the radioactive isotope¹²⁵ I, for example, by methods routinely practiced in the art. In aheterogeneous assay, e.g., the GST-target gene fusion protein can beanchored to glutathione-agarose beads. The interactive cellular orextracellular binding partner can then be added in the presence orabsence of the test compound in a manner that allows interaction andbinding to occur. At the end of the reaction period, unbound materialcan be washed away, and the labeled monoclonal antibody can be added tothe system and allowed to bind to the complexed components. Theinteraction between the target gene protein and the interactive cellularor extracellular binding partner can be detected by measuring the amountof radioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test compoundwill result in a decrease in measured radioactivity.

Alternatively, the GST-target gene fusion protein and the interactivecellular or extracellular binding partner can be mixed together inliquid in the absence of the solid glutathione-agarose beads. The testcompound can be added either during or after the species are allowed tointeract. This mixture can then be added to the glutathione-agarosebeads and unbound material is washed away. Again the extent ofinhibition of the target gene product/binding partner interaction can bedetected by adding the labeled antibody and measuring the radioactivityassociated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the target gene protein and/or the interactive cellular orextracellular binding partner (in cases where the binding partner is aprotein), in place of one or both of the full length proteins. Anynumber of methods routinely practiced in the art can be used to identifyand isolate the binding sites. These methods include, but are notlimited to, mutagenesis of the gene encoding one of the proteins andscreening for disruption of binding in a co-immunoprecipitation assay.Compensating mutations in the gene encoding the second species in thecomplex can then be selected. Sequence analysis of the genes encodingthe respective proteins will reveal the mutations that correspond to theregion of the protein involved in interactive binding. Alternatively,one protein can be anchored to a solid surface using methods describedin this Section above, and allowed to interact with and bind to itslabeled binding partner, which has been treated with a proteolyticenzyme, such as trypsin. After washing, a short, labeled peptidecomprising the binding domain may remain associated with the solidmaterial, which can be isolated and identified by amino acid sequencing.Also, once the gene coding for the for the cellular or extracellularbinding partner is obtained, short gene segments can be engineered toexpress peptide fragments of the protein, which can then be tested forbinding activity and purified or synthesized.

For example, and not by way of limitation, a target gene product can beanchored to a solid material as described, above, in this Section bymaking a GST-target gene fusion protein and allowing it to bind toglutathione agarose beads. The interactive cellular or extracellularbinding partner can be labeled with a radioactive isotope, such as ³⁵ S,and cleaved with a proteolytic enzyme such as trypsin. Cleavage productscan then be added to the anchored GST-target gene fusion protein andallowed to bind. After washing away unbound peptides, labeled boundmaterial, representing the cellular or extracellular binding partnerbinding domain, can be eluted, purified, and analyzed for amino acidsequence by well-known methods. Peptides so identified can be producedsynthetically or fused to appropriate facilitative proteins usingrecombinant DNA technology.

5.3.4. Assays for Amelioration of Body Weight Disorder Symptoms

Any of the binding compounds, including but not limited to, compoundssuch as those identified in the foregoing assay systems, may be testedfor the ability to ameliorate body weight disorder symptoms, which mayinclude, for example, obesity, anorexia, and/or an abnormal level offood intake. Cell-based and animal model-based assays for theidentification of compounds exhibiting such an ability to amelioratebody weight disorder symptoms are described below.

First, cell-based systems such as those described, above, in Section5.2.4.2, may be used to identify compounds which may act to amelioratebody weight disorder symptoms. For example, such cell systems may beexposed to a compound, suspected of exhibiting an ability to amelioratebody weight disorder symptoms, at a sufficient concentration and for atime sufficient to elicit such an amelioration of body weight disordersymptoms in the exposed cells. After exposure, the cells are examined todetermine whether one or more of the body weight disorder-like cellularphenotypes has been altered to resemble a more normal or more wild type,non-body weight disorder phenotype, or a phenotype more likely toproduce a lower incidence or severity of disorder symptoms.

In addition, animal-based body weight disorder systems, such as thosedescribed, above, in Section 5.2.4.1, may be used to identify compoundscapable of ameliorating body weight disorder-like symptoms. Such animalmodels may be used as test substrates for the identification of drugs,pharmaceuticals, therapies and interventions which may be effective intreating such disorders. For example, animal models may be exposed to acompound, suspected of exhibiting an ability to ameliorate body weightdisorder symptoms, at a sufficient concentration and for a timesufficient to elicit such an amelioration of body weight disordersymptoms in the exposed animals. The response of the animals to theexposure may be monitored by assessing the reversal of disordersassociated with body weight disorders such as obesity.

With regard to intervention, any treatments which reverse any aspect ofbody weight disorder-like symptoms should be considered as candidatesfor human body weight disorder therapeutic intervention. Dosages of testagents may be determined by deriving dose-response curves, as discussedin Section 5.6.1, below.

Gene expression patterns may be utilized in conjunction with eithercell-based or animal-based systems to assess the ability of a compoundto ameliorate body weight disorder-like symptoms. For example, theexpression pattern of one or more fingerprint genes may form part of afingerprint profile which may be then be used in such an assessment.Fingerprint profiles are described, below, in Section 5.7.1. Fingerprintprofiles may be characterized for known states, either body weightdisorder or normal states, within the cell- and/or animal-based modelsystems. Subsequently, these known fingerprint profiles may be comparedto ascertain the effect a test compound has to modify such fingerprintprofiles, and to cause the profile to more closely resemble that of amore desirable fingerprint. For example, administration of a compoundmay cause the fingerprint profile of a body weight disorder model systemto more closely resemble the control system. Administration of acompound may, alternatively, cause the fingerprint profile of a controlsystem to begin to mimic a body weight disorder state, which may, forexample, be used in further characterizing the compound of interest, ormay be used in the generation of additional animal models.

5.4. Compounds and Methods for Treatment of Body Weight Disorders

Described below are methods and compositions whereby body weightdisorder symptoms may be ameliorated. It is possible that body weightdisorders may be brought about, at least in part, by an abnormal levelof target gene product, or by the presence of a target gene productexhibiting an abnormal activity. As such, the reduction in the leveland/or activity of such target gene products would bring about theamelioration of body weight disorder-like symptoms. Techniques for thereduction of target gene expression levels or target gene productactivity levels are discussed in Section 5.4.1, below.

Alternatively, it is possible that body weight disorders may be broughtabout, at least in part, by the absence or reduction of the level oftarget gene expression, or a reduction in the level of a target geneproduct's activity. As such, an increase in the level of target geneexpression and/or the activity of such gene products would bring aboutthe amelioration of body weight disorder-like symptoms. Techniques forincreasing target gene expression levels or target gene product activitylevels are discussed in Section 5.4.2, below.

5.4.1. Compounds the Inhibit Expression, Synthesis or Activity of MutantTarget Gene Activity

As discussed above, target genes involved in body weight disorders maycause such disorders via an increased level of target gene activity. Avariety of techniques may be utilized to inhibit the expression,synthesis, or activity of such target genes and/or proteins.

For example, compounds such as those identified through assaysdescribed, above, in Section 5.3, which exhibit inhibitory activity, maybe used in accordance with the invention to ameliorate body weightdisorder symptoms. As discussed in Section 5.3, above, such moleculesmay include, but are not limited to, peptides, (such as, for example,peptides representing soluble extracellular portions of target geneproduct transmembrane receptors), phosphopeptides, small organic orinorganic molecules, or antibodies (including, for example, polyclonal,monoclonal, humanized, anti-idiotypic, chimeric or single chainantibodies, and FAb, F(ab')₂ and FAb expression library fragments, andepitope-binding fragments thereof). Techniques for determination ofeffective doses and administration of such compounds are describedbelow, in Section 5.6.1. Inhibitory antibody techniques are furtherdescribed, below, in Section 5.4.1.2.

Further, antisense and ribozyme molecules which inhibit expression ofthe target gene may also be used in accordance with the invention toinhibit the aberrant target gene activity. Such techniques aredescribed, below, in Section 5.4.1.1. Still further, as described,below, in Section 5.4.1.1, triple helix molecules may be utilized ininhibiting the aberrant target gene activity.

5.4.1.1. Inhibitory Antisense, Ribozyme and Triple Helix Approaches

Among the compounds which may exhibit the ability to ameliorate bodyweight disorder symptoms are antisense, ribozyme, and triple helixmolecules. Such molecules may be designed to reduce or inhibit eitherwild type, or if appropriate, mutant target gene activity. Techniquesfor the production and use of such molecules are well known to those ofskill in the art.

Anti-sense RNA and DNA molecules act to directly block the translationof mRNA by hybridizing to targeted mRNA and preventing proteintranslation. With respect to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between the -10 and+10 regions of the target gene nucleotide sequence of interest, arepreferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. (For a review, see Rossi, J., 1994, Current Biology4:469-471). The mechanism of ribozyme action involves sequence specifichybridization of the ribozyme molecule to complementary target RNA,followed by a endonucleolytic cleavage. The composition of ribozymemolecules must include one or more sequences complementary to the targetgene mRNA, and must include the well known catalytic sequenceresponsible for mRNA cleavage. For this sequence, see U.S. Pat. No.5,093,246, which is incorporated by reference herein in its entirety. Assuch, within the scope of the invention are engineered hammerhead motifribozyme molecules that specifically and efficiently catalyzeendonucleolytic cleavage of RNA sequences encoding target gene proteins.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the molecule of interest for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures, such as secondary structure, that may render theoligonucleotide sequence unsuitable. The suitability of candidatesequences may also be evaluated by testing their accessibility tohybridize with complementary oligonucleotides, using ribonucleaseprotection assays.

Nucleic acid molecules to be used in triplex helix formation for theinhibition of transcription should be single stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides must bedesigned to promote triple helix formation via Hoogsteen base pairingrules, which generally require sizeable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGC⁺triplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, contain a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in GGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called "switchback"nucleic acid molecule. Switchback molecules are synthesized in analternating 5'-3', 3'-5' manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

In instances wherein the antisense, ribozyme, and/or triple helixmolecules described herein are utilized to inhibit mutant geneexpression, it is possible that the technique may so efficiently reduceor inhibit the transcription (triple helix) and/or translation(antisense, ribozyme) of mRNA produced by normal target gene allelesthat the possibility may arise wherein the concentration of normaltarget gene product present may be lower than is necessary for a normalphenotype. In such cases, to ensure that substantially normal levels oftarget gene activity are maintained, therefore, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity may, be introduced into cells via gene therapymethods such as those described, below, in Section 5.5. that do notcontain sequences susceptible to whatever antisense, ribozyme, or triplehelix treatments are being utilized. Alternatively, in instances wherebythe target gene encodes an extracellular protein, it may be preferableto coadminister normal target gene protein in order to maintain therequisite level of target gene activity.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various well-known modifications to the DNA molecules may be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include but are not limited to, the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5' and/or 3' ends of themolecule or the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

5.4.1.2. Antibodies for Inhibition of Target Gene Products

Antibodies that are both specific for target gene protein and interferewith its activity may be used to inhibit target gene function. Wheredesirable, antibodies specific for mutant target protein which interferewith the activity of such mutant target product may also be used toinhibit target gene function. Such antibodies may be generated usingstandard techniques described in Section 5.2.3., supra, against theproteins themselves or against peptides corresponding to portions of theproteins. The antibodies include but are not limited to polyclonal,monoclonal, Fab fragments, single chain antibodies, chimeric antibodies,etc.

In instances where the target gene protein is intracellular and wholeantibodies are used, internalizing antibodies may be preferred. However,lipofectin or liposomes may be used to deliver the antibody or afragment of the Fab region which binds to the target gene productepitope into cells. Where fragments of the antibody are used, thesmallest inhibitory fragment which binds to the target protein's bindingdomain is preferred. For example, peptides having an amino acid sequencecorresponding to the domain of the variable region of the antibody thatbinds to the target gene protein may be used. Such peptides may besynthesized chemically or produced via recombinant DNA technology usingmethods well known in the art (e.g., see Creighton, 1983, supra; andSambrook et al., 1989, supra).

Alternatively, single chain neutralizing antibodies which bind tointracellular target gene product epitopes may also be administered.Such single chain antibodies may be administered, for example, byexpressing nucleotide sequences encoding single-chain antibodies withinthe target cell population by utilizing, for example, techniques such asthose described in Marasco et al. (Marasco, W. et al., 1993, Proc. Natl.Acad. Sci. USA 90:7889-7893).

In instances where the target gene protein is extracellular, or is atransmembrane protein, any of the administration techniques described,below in Section 5.6 which are appropriate for peptide administrationmay be utilized to effectively administer inhibitory target geneantibodies to their site of action.

5.4.2. Methods for Restoring Target Gene Activity

Target genes that cause body weight disorders may be underexpressedwithin body weight disorder situations. Alternatively, the activity oftarget gene products may be diminished, leading to the development ofbody weight disorder symptoms. Described in this Section are methodswhereby the level of target gene activity may be increased to levelswherein body weight disorder symptoms are ameliorated. The level of geneactivity may be increased, for example, by either increasing the levelof target gene product present or by increasing the level of activetarget gene product which is present.

For example, a target gene protein, at a level sufficient to amelioratebody weight disorder symptoms may be administered to a patientexhibiting such symptoms. Any of the techniques discussed, below, inSection 5.6, may be utilized for such administration. One of skill inthe art will readily know how to determine the concentration ofeffective, non-toxic doses of the normal target gene protein, utilizingtechniques such as those described, below, in Section 5 5.6.1.

Further, patients may be treated by gene replacement therapy. One ormore copies of a normal target gene or a portion of the gene thatdirects the production of a normal target gene protein with target genefunction, may be inserted into cells, using vectors which include, butare not limited to adenovirus, adeno-associated virus, and retrovirusvectors, in addition to other particles that introduce DNA into cells,such as liposomes. Additionally, techniques such as those describedabove may be utilized for the introduction of normal target genesequences into human cells.

Cells, preferably, autologous cells, containing normal target geneexpressing gene sequences may then be introduced or reintroduced intothe patient at positions which allow for the amelioration of body weightdisorder symptoms. Such cell replacement techniques may be preferred,for example, when the target gene product is a secreted, extracellulargene product.

Additionally, antibodies may be administered which specifically bind toa target protein and, by binding, serve to, either directly orindirectly, activate the target protein function. Such antibodies caninclude, but are not limited to polyclonal, monoclonal, FAb fragments,single chain antibodies, chimeric antibodies and the like. Theantibodies may be generated using standard techniques such as thosedescribed, above, in Section 5.2.3., and may be generated against theprotein themselves or against proteins corresponding to portions of theproteins. The antibodies may be administered, for example, according tothe techniques described, above, in Section 5.4.1.2.

5.6. Pharmaceutical Preparations and Methods of Administration

The identified compounds, nucleic acid molecules and cells that affecttarget gene expression, synthesis and/or activity can be administered toa patient at therapeutically effective doses to treat or ameliorate bodyweight disorders. A therapeutically effective dose refers to that amountof the compound sufficient to result in amelioration of symptoms of bodyweight disorder, or alternatively, to that amount of a nucleic acidmolecule sufficient to express a concentration of gene product whichresults in the amelioration of such symptoms.

5.6.1. Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e,, the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

5.6.2. Formulations and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycollate); or wetting agents (e.g., sodium lauryl sulphate).The tablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e,g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolds injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

5.7. Diagnosis of Body Weight Disorder Abnormalities

A variety of methods may be employed for the diagnosis of body weightdisorders, predisposition to body weight disorders, for monitoring theefficacy of antibody weight disorder compounds during, for example,clinical trials and for monitoring patients undergoing clinicalevaluation for the treatment of such body weight disorders.

Such methods may, for example, utilize reagents such as the fingerprintgene nucleotide sequences described in Sections 5.1, and antibodiesdirected against differentially expressed and pathway gene peptides, asdescribed, above, in Sections 5.1.3 (peptides) and 5.2.3 (antibodies).Specifically, such reagents may be used, for example, for: (1) thedetection of the presence of target gene mutations, or the detection ofeither over- or under-expression of target gene mRNA relative to thenon-body weight disorder state; and (2) the detection of either an over-or an under-abundance of target gene product relative to the non-bodyweight disorder state.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specificfingerprint gene nucleic acid or anti-fingerprint gene antibody reagentdescribed herein, which may be conveniently used, e.g., in clinicalsettings, to diagnose patients exhibiting body weight disorderabnormalities.

Any cell type or tissue in which the fingerprint gene is expressed maybe utilized in the diagnostics described below.

Among the methods which can be utilized herein are methods formonitoring the efficacy of compounds in clinical trials for thetreatment of body weight disorders. Such compounds can, for example, becompounds such as those described, above, in Section 5.4. Such a methodcomprises detecting, in a patient sample, a gene transcript or geneproduct which is differentially expressed in a body weight disorderstate relative to its expression in a normal, or non-body weightdisorder, state.

Any of the nucleic acid detection techniques described, below, inSection 5.7.1 or any of the peptide detection described, below, inSection 5.7.2 can be used to detect the gene transcript or gene productwhich is differentially expressed in a body weight disorder relative toits expression in the normal, or non-immune disorder, state.

During clinical trials, for example, the expression of a singlefingerprint gene, or alternatively, a fingerprint pattern of a cellinvolved in a body weight disorder can be determined in the presence orabsence of the compound being tested. The efficacy of the compound canbe followed by comparing the expression data obtained to thecorresponding known expression patterns in a normal, non-body weightdisorder state. Compounds exhibiting efficacy are those which alter thesingle fingerprint gene expression and/or the fingerprint pattern tomore closely resemble that of the normal, non-body weight disorderstate.

The detection of the product or products of genes differentiallyexpressed in a body weight disorder state relative to their expressionin a normal, or non-body weight disorder, state can also be used formonitoring the efficacy of potential anti-body weight disorder compoundsduring clinical trials. During clinical trials, for example, the leveland/or activity of the products of one or more such differentiallyexpressed genes can be determined in relevant cells and/or tissues inthe presence or absence of the compound being tested. The efficacy ofthe compound can be followed by comparing the protein level and/oractivity data obtained to the corresponding known levels/activities forthe cells and/or tissues in a normal, non-body weight disorder state.Compounds exhibiting efficacy are those which alter the pattern of thecell and/or tissue involved in the body weight disorder to more closelyresemble that of the normal, non-body weight disorder state.

5.7.1. Detection of Fingerprint Gene Nucleic Acids

DNA or RNA from the cell type or tissue to be analyzed may easily beisolated using procedures which are well known to those in the art.Diagnostic procedures may also be performed "in situ" directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents such as those described in Section 5.1may be used as probes and/or primers for such in situ procedures (see,for example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: ProtocolsAnd Applications", Raven Press, NY).

Fingerprint gene nucleotide sequences, either RNA or DNA, may, forexample, be used in hybridization or amplification assays of biologicalsamples to detect body weight disorder-related gene structures andexpression. Such assays may include, but are not limited to, Southern orNorthern analyses, single stranded conformational polymorphism analyses,in situ hybridization assays, and polymerase chain reaction analyses.Such analyses may reveal both quantitative aspects of the expressionpattern of the fingerprint gene, and qualitative aspects of thefingerprint gene expression and/or gene composition. That is, suchtechniques may include, for example, point mutations, insertions,deletions, chromosomal rearrangements, and/or activation or inactivationof gene expression.

Diagnostic methods for the detection of fingerprint gene-specificnucleic acid molecules may involve for example, contacting andincubating nucleic acids, derived from the cell type or tissue beinganalyzed, with one or more labeled nucleic acid reagents as aredescribed in Section 5.1, under conditions favorable for the specificannealing of these reagents to their complementary sequences within thenucleic acid molecule of interest. Preferably, the lengths of thesenucleic acid reagents are at least 15 to 30 nucleotides. Afterincubation, all non-annealed nucleic acids are removed from the nucleicacid:fingerprint molecule hybrid. The presence of nucleic acids from thecell type or tissue which have hybridized, if any such molecules exist,is then detected. Using such a detection scheme, the nucleic acid fromthe cell type or tissue of interest may be immobilized, for example, toa solid support such as a membrane, or a plastic surface such as that ona microtiter plate or polystyrene beads. In this case, after incubation,non-annealed, labeled nucleic acid reagents of the type described inSection 5.1 are easily removed. Detection of the remaining, annealed,labeled fingerprint nucleic acid reagents is accomplished using standardtechniques well-known to those in the art.

Alternative diagnostic methods for the detection of fingerprint genespecific nucleic acid molecules may involve their amplification, e.g.,by PCR (the experimental embodiment set forth in Mullis, K. B., 1987,U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, F., 1991, Proc.Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc.Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. etal., 1988, Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In one embodiment of such a detection scheme, a cDNA molecule isobtained from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). Cell types or tissues fromwhich such RNA may be isolated include any tissue in which wild typefingerprint gene is known to be expressed. A sequence within the cDNA isthen used as the template for a nucleic acid amplification reaction,such as a PCR amplification reaction, or the like. The nucleic acidreagents used as synthesis initiation reagents (e.g., primers) in thereverse transcription and nucleic acid amplification steps of thismethod are chosen from among the fingerprint gene nucleic acid reagentsdescribed in Section 5.1. The preferred lengths of such nucleic acidreagents are at least 9-30 nucleotides. For detection of the amplifiedproduct, the nucleic acid amplification may be performed usingradioactively or non-radioactively labeled nucleotides. Alternatively,enough amplified product may be made such that the product may bevisualized by standard ethidium bromide staining or by utilizing anyother suitable nucleic acid staining method.

In addition to methods which focus primarily on the detection of onefingerprint nucleic acid sequence, fingerprint patterns or profiles mayalso be assessed in such detection schemes. "Fingerprint pattern" or"fingerprint profile", as used herein, refers to the pattern of mRNAexpression obtained for a given tissue or cell type under a given set ofconditions, and includes the mRNA expression of at least two of thegenes within the tissue or cell type. Such conditions may include, butare not limited to body weight disorders, including obesity, andconditions relevant to processes involved in body weight or appetiteregulation, including any of the control or experimental conditionsdescribed in the paradigms of Section 5.1.1.1, above. Fingerprintprofiles may be generated, for example, by utilizing a differentialdisplay procedure, as discussed, above, in Section 5.1.1.2, Northernanalysis and/or RT-PCR. Any of the gene sequences described, above, inSection 5.2.1 may be used as probes and/or PCR primers for thegeneration and corroboration of such fingerprint profiles.

5.7.2. Detection of Target Gene Peptides

Antibodies directed against wild type or mutant fingerprint genepeptides, which are discussed, above, in Section 5.2.3, may also be usedas body weight disorder diagnostics and prognostics, as described, forexample, herein. Such diagnostic methods, may be used to detectabnormalities in the level of fingerprint gene protein expression, orabnormalities in the structure and/or temporal, tissue, cellular, orsubcellular location of fingerprint gene protein. Structural differencesmay include, for example, differences in the size, electronegativity, orantigenicity of the mutant fingerprint gene protein relative to thenormal fingerprint gene protein.

Protein from the tissue or cell type to be analyzed may easily beisolated using techniques which are well known to those of skill in theart. The protein isolation methods employed herein may, for example, besuch as those described in Harlow and Lane (Harlow, E. and Lane, D.,1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York), which is incorporated herein byreference in its entirety.

Preferred diagnostic methods for the detection of wild type or mutantfingerprint gene peptide molecules may involve, for example,immunoassays wherein fingerprint gene peptides are detected by theirinteraction with an anti-fingerprint gene product- specific antibody.

For example, antibodies, or fragments of antibodies, such as thosedescribed, above, in Section 5.2.3, useful in the present invention maybe used to quantitatively or qualitatively detect the presence of wildtype or mutant fingerprint gene peptides. This can be accomplished, forexample, by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below, this Section) coupled with lightmicroscopic, flow cytometric, or fluorimetric detection. Such techniquesare especially preferred if the fingerprint gene peptides are expressedon the cell surface.

The antibodies (or fragments thereof) useful in the present inventionmay, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of fingerprint genepeptides. In situ detection may be accomplished by removing ahistological specimen from a patient, and applying thereto a labeledantibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the fingerprint gene peptides, butalso their distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays for wild type or mutant fingerprint gene peptides typicallycomprise incubating a biological sample, such as a biological fluid, atissue extract, freshly harvested cells, or cells which have beenincubated in tissue culture, in the presence of a detectably labeledantibody capable of identifying fingerprint gene peptides, and detectingthe bound antibody by any of a number of techniques well-known in theart.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled fingerprint genespecific antibody. The solid phase support may then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on solid support may then be detected by conventional means.

By "solid phase support or carrier" is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-wild type or anti-mutantfingerprint gene product antibody may be determined according to wellknown methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

One of the ways in which the fingerprint gene peptide-specific antibodycan be detectably labeled is by linking the same to an enzyme and use inan enzyme immunoassay (EIA) (Voller, A., "The Enzyme LinkedImmunosorbent Assay (ELISA)" 1978 Diagnostic Horizons 2:1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E.,1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, ENZYMEIMMUNOASSAY, CRC Press, Boca Raton, Fla.; Ishikawa, E. et al., (eds.),1981, ENZYME IMMUNOASSAY, Kgaku Shoin, Tokyo). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵² Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

6. Example: Identification and Characterization of an Obesity-RelatedGene

In the Example presented in this Section, one of the paradigmsdescribed, above, in Section 5.1.1.1, the genetic obesity paradigm, isutilized to identify a gene which is not only differentially expressedin genetically obese test animals, but is identical to a gene which haspreviously been implicated in processes involved in body weightregulation. Thus, the successful identification, here, of this genecorroborates the usefulness of the paradigm approach of the inventionfor the identification of genes involved in body weight disorders and/orin body weight or appetite regulation.

6.1. Materials and Methods

Genetic Obesity paradigms: 15 female C57B1/6J ob/ob mice and leanlittermate controls (15 female C57B1/6J ?/+) and 15 male C57B1/Ks db/dbmice and lean littermate controls (15 male C57B1/ks +/+) were receivedfrom Jackson labs at 4.5 weeks of age, and housed individually on normalmouse chow (West, D. B., 1992, Am. J. Physiol. 262:R1025-R1032) for 1week prior to the initiation of the study. The four groups of 15 miceeach were then sacrificed by CO₂ euthanasia and tissues were collected.Body weight (grams) of the four groups of mice at the time of sacrificewas measured.

Tissue collection and RNA isolation: Following CO₂ asphyxiation, tissueswere removed and quick frozen on dry ice. Samples within an experimentalor control group (15 animals per group) were then homogenized togetherwith a mortar and pestle under liquid nitrogen.

Total cellular RNA was extracted from tissue with either RNAzol™ orRNAzolB™ (Tel-Test, Friendswood, Tex.), according to the manufacturer'sinstructions. Briefly, the tissue was solubilized in an appropriateamount of RNAzol™ or RNAzolB™, and RNA was extracted by the addition of1/10 v/v chloroform to the solubilized sample followed by vigorousshaking for approximately 15 seconds. The mixture was then centrifugedfor 15 minutes at 12,000 g and the aqueous phase was removed to a freshtube. RNA was precipitated with isopropanol. The resultant RNA pelletwas dissolved in water and re-extracted with an equal volume ofchloroform to remove any remaining phenol. The extracted volume wasprecipitated with 2 volumes of ethanol in the presence of 150 mM sodiumacetate. The precipitated RNA was dissolved in water and theconcentration determined spectroscopically (A₂₆₀).

Differential display: Total cellular RNA (10-50 μg) was treated with 20Units DNase I (Boehringer Mannheim, Germany) in the presence of 40 Unitsribonuclease inhibitor (Boehringer Mannheim, Germany). After extractionwith phenol/chloroform and ethanol precipitation, the RNA was dissolvedin DEPC (diethyl pyrocarbonate)-treated water.

Differential mRNA display was carried out as described, above, inSection 5.1.1.2. RNA (0.4-2 μg) was reverse-transcribed usingSuperscript reverse transcriptase (GIBCO/BRL). The cDNAs were thenamplified by PCR on a Perkin-Elmer 9600 thermal cycler. The reactionmixtures (20 μl) included arbitrary decanucleotides and one of twelvepossible T₁₁ VN sequences, wherein V represents either dG, dC, or dA,and N represents either dG, dT, dA, or dC. Parameters for the 40 cyclePCR were as follows: Hold 94° C. 2 minutes; Cycle (40 rounds) 94° C. 15seconds, 40° C. 2 minutes; Ramp to 72° 30 seconds; Hold 72° C. 5minutes; Hold 4° C.

Radiolabelled PCR amplification products were analyzed byelectrophoresis on 6% denaturing polyacrylamide gels.

Reamplification and subcloning: PCR bands of interest were recoveredfrom sequencing gels and reamplified.

Briefly, autoradiograms were aligned with the dried gel, and the regioncontaining the bands of interest was excised with a scalpel. The excisedgel fragment was eluted by soaking in 100 μl TE (Tris-EDTA) buffer atapproximately 100° C. for 15 minutes. The gel slice was then pelleted bybrief centrifugation and the supernatant was transferred to a newmicrocentrifuge tube. DNA was combined with ethanol in the presence of100 mM Sodium acetate and 30 μg glycogen (Boerhinger Mannhein, Germany)and precipitated on dry ice for approximately 10 minutes. Samples werecentrifuged for 10 minutes and pellets were washed with 80% ethanol.Pellets were resuspended in 10 μl distilled water.

5 μl of the eluted DNA were reamplified in a 100 μl reaction containing:standard Cetus Taq polymerase buffer, 20 μM dNTPs, 1 μM of each of theoligonucleotide primers used in the initial generation of the amplifiedDNA. Cycling conditions used were the same as the initial conditionsused to generate the amplified band, as described above. One-half of theamplification reaction was run on a 2% agarose gel and eluted usingDE-81 paper (Whatman Paper, Ltd., England) as described in Sambrook etal., supra. Recovered fragments were ligated into the cloning vectorpCR™II (Invitrogen, Inc., San Diego Calif.) and transformed intocompetent E. coli strain DH5α (Gibco/BRL, Gaithersburg, Md.). Colonieswere grown on LB-agar plates containing ampicillin (100 μg/ml) and X-gal(40 μg/ml) to permit blue/white selection.

Sequence analysis: After subcloning, reamplified cDNA fragments weresequenced on an Applied Biosystems Automated Sequencer (AppliedBiosystems, Inc. Seattle, Wash.). Sequence was obtained from fourindependent transformants containing the same insert. The nucleotidesequence shown herein represents the consensus of the informationobtained from the four sequences. Such primary sequence data was editedand trimmed of vector sequences and highly repetitive sequences and usedto search Genbank databases using the BLAST (Altschul, S. F. et al.,1990, J. Mol. Biol. 215:403-410) program.

Northern analysis: RNA samples were electrophoresed in a denaturingagarose gel containing 1-1.5% agarose (SeaKem™ LE, FMC BioProducts,Rockland, Me.) containing 6.3% formaldehyde. Samples containing 5-20 μgof total RNA were mixed with denaturing loading solution (72% deionizedformamide and bromophenol blue) and heated to 70° C. for 5 minutes.Samples were placed on ice and immediately loaded onto gels. Gels wererun in 1× MOPS buffer (100 mM MOPS, 25 mM sodium acetate, 5 mM EDTA).After electrophoresis, the gels were stained with ethidium bromide andvisualized with ultraviolet light.

After completion of electrophoresis, gels were soaked in 50 mM sodiumhydroxide with gentle agitation for approximately 30 minutes to lightlycleave RNA. Gels were rinsed twice in water and then neutralized bysoaking in 0.1M Tris-HCl (pH 7.5) for approximately 30 minutes. Gelswere briefly equilibrated with 20× SSC (3M sodium chloride, 0.3M sodiumcitrate) and then transferred to nylon membranes such as Hybond™,-N,(Amersham, Inc., Arlington Heights, Ill.) or Zeta-Probe (Bio-Rad, Inc.,Hercules, Calif.) overnight in 20× SSC. Membranes containing transferredRNA were baked at 80° C. for 2 hours to immobilize the RNA.

DNA fragments to be used as probes were of various sizes and werelabeled using a random hexamer labeling technique. Briefly, 25 ng of apurified DNA fragment was used to generate each probe. Fragments wereadded to a 20 μl random hexanucleotide labeling reaction (BoehringerMannhein, Inc., Indianapolis, Ind.) containing random hexamers and a mixof the nucleotides dCTP, dGTP, and dTTP (at a final concentration of 25μM each). The reaction mix was heat-denatured at 100° C. for 10 minutesand then chilled on ice. 5 μl of α-³² P-dATP (50 μCi; Amersham, Inc.,Arlington Heights, Ill.) and Klenow DNA polymerase (2 units; BoehringerMannheim, Inc., Indianapolis, Ind.) were added. Reactions were incubatedat 37° for 30 minutes. Following incubation, 30 μl water was added tothe labeling reaction and unincorporated nucleotides were removed bypassing the reactions through a BioSpin-6™ chromatography column(Bio-Rad, Inc., Hercules, Calif.). Specific incorporation was determinedusing a scintillation counter. 1-5×10⁶ cpm were used per mlhybridization mixture.

Nylon membranes containing immobilized RNA were prehybridized accordingto manufacturer's instructions. Radiolabelled probes were heat denaturedat 70° C. in 50% deionized formamide for 10 minutes and ten added to thehybridization mixture (containing 50% formamide, 10% dextran sulfate,0.1% SDS, 100 μg/ml sheared salmon sperm DNA, 5× SSC, 5× Denhardt'ssolution, 30 mM Tris-HCl (pH 8.5), 50 mM NaPO₄ (pH 6.5). Hybridizationswere carried out at 42° C. overnight. Nylon membranes were then bathedfor 2 minutes in a wash solution of 0.2× SSC and 0.1% SDS at roomtemperature to remove most of the remaining hybridization solution. Themembranes were then bathed twice in fresh 42° C. preheated wash solutionfor 20 minutes. Filters were covered in plastic wrap and exposed toautoradiographic film to visualize results.

6.2. Results

Genetic obesity paradigms were utilized to identify genes which aredifferentially expressed in obese versus lean mice. Specifically, ob/oband db/db obese mice were utilized in conjunction with lean littermatecontrol mice, as described, above, in Section 6.1.

RNA samples isolated from liver tissue of the ob/ob, db/db, andlittermate control mice were analyzed via differential displaytechniques. FIG. 1 shows amplified fragments obtained from these tissueswhen subjected to PCR with 11 separate primer pair combinations. Thearrow in FIG. 1, indicates a PCR product, designated band L36, which wasjudged to be differentially expressed among the lean and obese (ob anddb) samples, with a larger amount of expression in the obese relative tothe lean control samples.

To confirm the putative differential gene regulation, the amplified L36band was recovered, reamplified, and used to probe Northern RNA blotswhich were prepared with the original liver RNA samples. FIG. 2 showsthe results of one such Northern blot analysis, in which the steadymessages corresponding to cDNA band L36 are shown to be significantlyincreased in RNA samples derived from both ob/ob and db/db mice comparedto lean littermate controls. Thus, this study confirmed the putativedifferential regulation which had been suggested by the differentialdisplay result.

The reamplified fragment corresponding to band L36 was subcloned into acloning vector and sequenced, as described, above, in Section 6.1.Plasmid DNA from four independent transformants was sequenced. All fourplasmids were shown to contain the same insert and a consensus sequenceof the four sequences was compiled and is shown in FIG. 3A.

A database search with this consensus sequence resulted in an alignmentwith greater than 99% identity to a mouse stearoyl-Co-A desaturase gene,SCD1 (Ntambi, J. M. et al., 1988, J. Biol. Chem. 263:17291-17300;Kaestner, K. H. et al., 1989, J. Biol. Chem. 264:14755-14761), whichencodes an enzyme that converts saturated fats to mono-unsaturated fatsin the liver (FIG. 3B).

Mouse stearoyl-Co-A-desaturase mRNA is induced in liver upon feeding offasted animals (Ntambi, J. M. et al., supra; Ntambi, J. M., 1992, J.Biol. Chem. 267:10925-10930). Further, in studies of lean versus obesemice, rats and chickens, stearoyl-Co-A enzymatic activity hasconsistently been reported to be higher in fat than lean animals (Esner,M., 1979, Biochem. J. 180:551-558; Wahle, K. W. J. and Radcliffe, J. D.,1977, Lipids 12:135-139; Legrand, P. et al., 1987, Comp. Biochem.Physiol. 87B:789-792). Additionally, it has been shown thatstearoyl-Co-A activity is higher in chickens than turkeys (Kouba, M. etal., 1993, Comp. Biochem. Physiol. 105A:359-362). It is considered thatturkeys are a low fat animal as compared to chickens. Thus, it is likelythat the stearoyl-Co-A enzyme is involved in such body weight regulatingprocesses as control, metabolism and storage of dietary components.

Therefore, by utilizing the genetic obesity paradigms described in thisSection and in Section 5.1.1.1, above, a differentially regulated gene,the mouse stearoyl-Co-A gene, involved in body weight regulation hasbeen identified, thereby corroborating the usefulness of such paradigmsin identifying genes important to body weight disorders, and/or bodyweight or appetite regulation.

7. Example: Identification of Genes Differentially Expressed in Responseto Short Term Appetite Control Paradigms

In the Example presented in this Section, the short term appetitecontrol paradigm, as described, above, in Section 5.1.1.1, is utilizedto identify gene sequences which are differentially expressed and whichmay contribute to body weight disorders and/or may be involved in suchprocesses as body weight regulation or appetite modulation.

7.1. Materials and Methods

Short term appetite control paradigm: 45 male C57B1/6J mice 8 weeks ofage were received from Jackson labs. The animals were randomized intothree groups of 15 mice each, and housed individually on normal mousechow (West, D. B. et al., 1992, Am. J. Physiol. 262:R1025-R1032)) for 1week prior to the initiation of the study. Group 1 mice (Control) weremaintained on ad lib mouse chow up until the time of sacrifice. Group 2mice (Fasted) were fasted for 24 hours prior to sacrifice (watercontinuously available). Group 3 mice (Fasted-Refed) were fasted for 24hours and then offered a highly palatable meal (mouse chow mixed withpeanut butter) for 1 hour prior to sacrifice. All mice were weighedimmediately before the initiation of the experiment and immediatelyafterward. All mice were sacrificed by CO₂ asphyxiation.

RT-PCR analysis: Quantitative RT-PCR was performed as follows. 1-2 μg oftotal RNA, prepared as described, above, in Section 6.1, was reversetranscribed with oligo dT.sub.(12-18) primers and Superscript™ RNAaseH-reverse transcriptase (Gibco-BRL, Gaithersburg, Md.). Briefly, RNA wascombined with 1 μl oligo dT (500 μg/ml) in a total volume of 11 μl . Themixture was heated to 70° C. for 10 minutes and chilled on ice. After abrief centrifugation RNA was reverse transcribed for 1 hour. Aliquots ofthe first strand cDNA were stored at -20° C. until just prior to use.

Expression levels were determined by PCR amplification of serialdilutions of first strand cDNA. In this procedure, cDNA is seriallydiluted in water. The dilutions are then batch amplified by PCR usingsequence-specific primers. All PCR reactions are amplified underidentical conditions. Therefore, the amount of product generated shouldreflect the amount of sequence template which was initially present.5-10 fold dilutions of cDNA were used and enough dilutions were usedsuch that the amount of product subsequently produced ranged fromclearly visible, by UV illumination of ethidium bromide-stained gels, tobelow detection levels. The method described herein can distinguish10-fold differences 10 in expression levels.

Primers were designed for the amplification of the sequenced amplifiedbands, which were chosen using the program OLIGO (National Biosciences,Plymouth, Minn.). All quantitative PCR reactions were carried out in a9600 Perkin-Elmer PCR machine (Perkin-Elmer). Generally, amplificationconditions were as follows: 30-40 cycles consisting of a 95° C.denaturation for 30 seconds, 72° C. extension for 1 minute, 50°-60° C.annealing for 30 seconds. Following cycling, reactions were extended for10 minutes at 72° C.

Other procedures: All other tissue collection, RNA isolation,differential display, sequence analysis, and Northern proceduresperformed in the experiments described in this Section were asdescribed, above, in Section 6.1.

7.2. Results

Mice, as described, above, in Section 7.1, were utilized as part ofshort term appetite control paradigms. Briefly, C57B1/6J mice weredivided into Control, Fasted, and Fasted-Refed groups, in order toidentify genes which are differentially expressed in response to hungerand satiety.

The mice were weighed immediately before the initiation of the study andimmediately prior to their sacrifice at the end of the study. Bodyweights (in grams) were as in Table 3, below:

                  TABLE 3                                                         ______________________________________                                                Control   Fasted    Fasted-Refed                                      ______________________________________                                        Before Study                                                                            23.9+/-1.3  23.3+/-1.1                                                                              23.2+/-1.4                                    After Study                                                                             24.4+/-1.4  19.3+/-1.1                                                                              21.7+/1-1.4                                   ______________________________________                                    

Upon sacrifice, control, Fasted, and Fasted-Refed tissues were collectedand immediately frozen. The tissues collected were: hypothalamus, liver,small intestine, pancreas, stomach, and omental adipose tissue. RNA wascollected from the tissue samples obtained and was subjected todifferential display, as described, above, in Section 7.1.

Utilizing such short term appetite control paradigms and differentialdisplay techniques, several gene sequences were identified. Dataobtained from such sequences is summarized, below, in Table 4. Thedifferential expression data identifying these gene sequences ascorresponding to genes which may be involved in body weight disordersand/or body weight or appetite regulation is listed in the columnsheaded "Fasted" and "Refed", depending on the paradigm in whichdifferential expression of a given gene was analyzed. Further, thetissue in which the differential expression was observed is noted, as isthe difference in expression of each gene in the experimental (eitherfasted or refed animals) versus control tissues. "" indicates that geneexpression is increased (i.e., there is an increase in the amount ofdetectable mRNA produced by a given gene) in experimental versus controltissue, while ".arrow-down dbl." indicates that gene expression isdecreased (i.e., there is an decrease in the amount of detectable mRNAproduced by a given gene) in experimental versus control tissue. Table 4also notes whether the gene sequence corresponds to a gene which hadpreviously been identified, and additionally notes the figure in whichthe nucleotide sequence of the given sequence is listed.

                  TABLE 4                                                         ______________________________________                                                                      Previously                                      Gene      Fasted      Refed   Known  Sequence                                 ______________________________________                                        P3 (SEQ. ID                                                                             Pancreas        Yes      FIG. 4                                     NO:4, 39)                                                                     P13 (SEQ. ID                                                                            Pancreas        Yes      FIG. 5                                     NO:7,40-42)                                                                   F5 (SEQ. ID                                                                             Adipose         Yes      FIG. 6                                     NO:12)                                                                        L7/L21 (SEQ.                                                                            Liver           Yes      FIG. 9                                     ID NO:18)                                                                     H27 (SEQ. ID                                                                            .arrow-down dbl.Hypothalalmus                                                                 Yes       FIG. 20                                   NO:49)                                                                        ______________________________________                                    

In addition to the tissues, listed above in Table 4, in which theinitial differential expression was observed, further analysis of thetissue distribution of gene expression of the differentially expressedgenes has been conducted. Such an analysis consisted of either Northernor RT-PCR studies, or both, as described, above, in Section 7.1. Thetissue distribution information obtained for the above-listed genes isreported, above, in Table 1 of Section 5.2.1.

Database searches revealed that the genes listed in Table 4, above,identified via the short term appetite control paradigms describedherein have previously been identified. Specifically, P3 (SEQ ID NO.:4)represents the gene encoding mouse glutamine synthetase (FIG. 4); P13(SEQ ID NO.:7, 40-42) represents the gene encoding mouse isletregenerating protein (FIG. 5); F5 (SEQ ID NO.:12) represents the geneencoding mouse α-amylase (FIG. 6); L7/L21 (SEQ ID NO.:18) represents thegene encoding mouse cytochrome c oxidase subunit I (FIG. 9); and H27(SEQ ID NO:49) represents the gene encoding mouse autoantigen La (FIG.20).

8. Example: Identification of Genes Differentially Expressed in Responseto Genetic Obesity Paradigms

In the Example presented in this Section, genetic obesity paradigms, asdescribed, above, in Section 5.1.1.1, were utilized to identify genesequences which are differentially expressed and which may contribute tobody weight disorders and/or may be involved in such processes as bodyweight regulation or appetite modulation.

8.1. Materials and Methods

Genetic obesity paradigms: Animals and animal treatments were asdescribed above, in Section 6.1.

Other procedures: All other tissue collection, RNA isolation,differential display and sequence analysis procedures performed in theexperiments described in this Section were as described, above, inSection 6.1. RT-PCR procedures were as described, above, in Section 7.1.

8.2. Results

Ob/ob, db/db, and lean littermate control mice, as described, above, inSection 8.1, were utilized as part of genetic obesity paradigms. Themice were weighed at the end of the study, immediately prior tosacrifice.

Upon sacrifice, tissues were collected from the four groups (i.e.,ob/ob, db/db and lean littermate controls) and immediately frozen. Thetissues collected were: hypothalamus, liver, small intestine, pancreas,stomach, epididymal or uterine fat pads, and skeletal muscle. RNA wascollected from the tissue samples obtained and was subjected todifferential display, as described, above, in Section 8.1.

Utilizing such genetic obesity paradigms and differential displaytechniques, several gene sequences, corresponding to both unique (i.e.,previously unknown) and known genes were identified. Data obtained fromsuch sequences is summarized, below, in Table 5. The differentialexpression data identifying these gene sequences as corresponding togenes which may be involved in body weight disorders and/or body weightor appetite regulation is listed in the columns headed "Ob/ob and"Db/db", depending on the paradigm in which differential expression of agiven gene was analyzed. Further, the tissue in which the differentialexpression was observed is noted, as is the difference in expression ofeach gene in the experimental (either ob or db animals) versus controltissues. "" indicates that gene expression is increased (i.e., there isan increase in the amount of detectable mRNA produced by a given gene)in experimental versus control tissue, while ".arrow-down dbl."indicates that gene expression is decreased (i.e., there is an decreasein the amount of detectable mRNA produced by a given gene) inexperimental versus control tissue. Further, "+" indicates that geneexpression was activated in experimental versus control tissue, i.e.,mRNA was detectable in experimental tissue whereas none was detectablein control tissue.

Table 5 also notes whether the gene sequence corresponds to a gene whichhad previously been identified, and additionally notes in which figurethe nucleotide sequence of the given sequence is listed.

                  TABLE 5                                                         ______________________________________                                                                     Prev.                                            Gene     ob/ob     db/db     known  Seq.                                      ______________________________________                                        F49                +Adipose  No     FIG. 14                                   (SEQ. ID                                                                      NO:34)                                                                        murine   Adipose   Adipose   No     FIG. 16A,16B                              C5* (SEQ.                                                                     ID                                                                            NO:36)                                                                        L31/F74  .arrow-down dbl.Adipose                                                                 .arrow-down dbl.Adipose                                                                 Yes    FIG. 8A,8B                                SEQ. ID                                                                       NO:16)                                                                        L7/L21   Liver     Liver     Yes    FIG. 9                                    (SEQ. ID                                                                      NO:18)                                                                        L29      .arrow-down dbl.Liver                                                                             Yes    FIG. 10                                   (SEQ. ID                                                                      NO:20)                                                                        L38      Liver     Liver     Yes    FIG. 11                                   (SEQ. ID                                                                      NO:22,43)                                                                     L37      Liver               Yes    FIG. 12                                   (SEQ. ID                                                                      NO:25,44-45)                                                                  L34 (SEQ Liver               Yes    FIG. 22                                   ID NO:57)                                                                     ______________________________________                                         *The mouse C5 sequence was first identified via sequence homology. C5 was     then subsequently tested in ob and db mice, at which time it was              identified to represent a differentially expressed gene sequence.        

In addition to the tissues, listed above in Table 5, in which theinitial differential expression was observed, further analysis of thetissue distribution of gene expression of the differentially expressedgenes has been conducted. Such an analysis consisted of either Northernor RT-PCR studies, or both, as described, above, in Section 8.1. Thetissue distribution information obtained for the above-listed genes isreported, above, in Table 1 of Section 5.2.1.

As described above, several of the gene sequences identified via thegenetic obesity paradigms of the invention represent previously unknowngenes. These include F49 (SEQ ID NO.:34) and C5 (SEQ ID NO.:36).

A putative full length cDNA clone (FIG. 14; SEQ ID NO.:34) correspondingto the entire coding sequence of the fat-specific F49 gene has beenisolated utilizing the techniques described, above, in Section 5.1.1.2.Hybridization of F49 nucleotide sequences to genomic DNA of severaldivergent organisms reveals that the F49 gene is conserved in mostmammals, including monkeys and humans, while the gene appears to beabsent from chicken and yeast.

The F49 coding sequence predicts a 96 amino acid protein (SEQ IDNO.:35), shown in FIG. 14. The sequence strongly suggests that the F49gene product is a secreted protein. Take, for example, the F49 geneproduct hydropathy plot depicted in FIG. 15. The strongly hydrophobicamino-terminal portion of the amino acid sequence is highly suggestiveof a signal sequence characteristic of secreted proteins.

A putative full-length cDNA clone (FIGS. 16A, 16B; SEQ ID NO.:36)corresponding to the entire coding sequence of the murine C5 gene hasbeen isolated utilizing the techniques described, above, in Section5.1.1.2. The C5 coding sequence predicts the protein whose amino acidsequence is shown in FIGS. 16A, 16B (SEQ ID NO.:37) This amino acidsequence bears a 50% identity to the mouse brown fat uncoupling protein,and thus represents a newly discovered brown fat uncoupling homologue.In order to further characterize the C5 gene product, a C5 cDNA has beencloned into yeast and mammalian (pcDNA3; Invitrogen) expression vectorsto test whether the C5 gene product exhibits uncoupling activity.

Additionally, database searches have revealed that several of the genesidentified via the genetic obesity paradigms described herein havepreviously been identified (see Table 5). For example, L31/F74 (SEQ IDNO.:16) represents the gene encoding the mouse major urinary protein II(FIGS. 8A, 8B); L7/L21 (SEQ ID NO.:18) represents the gene encodingmouse cytochrome c oxidase subunit I (FIG. 9); L29 (SEQ ID NO.:20)represents the gene encoding mouse testosterone 15-α hydroxylase (FIG.10); L38 (SEQ ID NO.:22, 43) represents the gene encoding mouse 24p3, alipocalin family member of unknown function (FIG. 11); L37 (SEQ IDNO.:25, 44-45) represents the gene encoding mouse p6-5, a gene which is86% homologous to rat preproelastase I (FIG. 12); L34 (SEQ ID NO:57;FIG. 22) represents the mouse homolog of a human gene, which will bereferred to herein as the "human L34 gene". The human L34 gene, whosesequence can be found in Hou et al. (Hod et al., 1994, In Vitro CellDev. Biol. Anim. 30A:111-114, which is incorporated herein by referencein its entirety) was cloned from a hepatoblastoma cell line and encodesa leucine-rich transmembrane protein. The mouse L34 gene exhibits an 82%identity with the human L34 gene at the nucleotide level, and thederived amino acid sequence of the mouse L34 gene product exhibits an86% identity with the human L34 gene product.

Several of the previously identified genes which these studies havedemonstrated to be differentially expressed in obese versus lean controlsubjects have never before been associated with processes involved inbody weight regulation, appetite regulation, or body weight disorders,such as obesity. Among these genes are the genes encoding the mousemajor urinary protein II (L31/F74), mouse testosterone 15-α hydroxylase(L29), mouse 24p3 (L38), mouse p6-5 (L37), and human and mouse L34proteins.

9. Example: Identification of Genes Differentially Expressed in Responseto Set Point Paradigms

In the Example presented in this Section, set point paradigms, asdescribed, above, in Section 5.1.1.1, were utilized to identify genesequences which are differentially expressed and which may contribute tobody weight disorders and/or may be involved in such processes as bodyweight regulation or appetite modulation.

9.1. Materials and Methods

Set point paradigms: 45 male C57B1/6J mice 8 weeks of age were receivedfrom Jackson labs. The animals were randomized into 3 groups of 15 miceeach, and housed individually on normal mouse chow for 1 week prior tothe initiation of the study. Group 1 mice (Control) were maintained onad lib mouse chow for an additional five days in order to calculate thedaily food intake. Group 2 mice (underweight) then received a fractionof normal food intake (60-90%) so as to reduce and maintain their bodyweight at approximately 80% of control values. Group 3 mice (overweight)were given a cafeteria diet so as to bring their body weights to 125% ofcontrol. The three groups of 15 mice each were then sacrificed by CO₂euthanasia and tissues were immediately collected. Body weights of thethree groups of 15 mice were taken at the time of sacrifice.

Other Procedures: All other tissue collection, RNA isolation,differential display, sequence analysis, and Northern proceduresperformed in the experiments described in this Section were asdescribed, above, in Section 6.1. RT-PCR quantitative analysis wasperformed as described, above, in Section 7.1.

9.2. Results

Mice, as described, above, in Section 9.1, were utilized as part of setpoint paradigms. The mice were weighed at the end of the study,immediately prior to sacrifice.

Upon sacrifice, tissues were collected from the three groups (i.e.,Control, Underweight and Overweight) and immediately frozen. The tissuescollected were hypothalamus, liver, small intestine, pancreas, stomach,epididymal fat pads, and skeletal muscle. RNA was collected from thetissue samples obtained and was subjected to differential display, asdescribed, above, in Section 9.1.

Utilizing such set point paradigms and differential display techniques,gene sequences L57, F84 and L31/F74, corresponding to known genes, wereidentified, as summarized in Table 6, below. In addition to differentialexpression information, Table 6 also notes in which figure nucleotidesequences of the identified genes are listed.

                  TABLE 6                                                         ______________________________________                                                                      Previously                                      Gene      Overweight                                                                              Underweight                                                                             known  Seq.                                     ______________________________________                                        L57 (SEQ. ID    Liver     Yes      FIG. 13                                    NO:29,46-48)                                                                  F84 (SEQ. ID    .arrow-down dbl.Adipose                                                                 Yes      FIG. 21                                    NO:52,54)                                                                     L31/F74         .arrow-down dbl.Liver                                                                   Yes      FIG. 8A,8B                                 (SEQ. ID                                                                      NO:16)                                                                        ______________________________________                                    

Database searches have revealed that the L57 (SEQ ID NO: 29, 46-48) genesequence corresponds to a previously known gene, the gene encoding themouse orphan nuclear hormone receptor (FIG. 13) and the F84 (SEQ ID NO:53, 55) gene sequence encodes mouse cytochrome p450 IID (FIG. 21). Asdiscussed, above, in Section 8.2, the L31/F74 gene sequence encodes themouse major urinary protein II. Interestingly, these gene products havenever before been associated with processes involving body weight orappetite regulation or body weight disorders.

10. Example: Isolation and Characterization of the Human C5 Gene

Described in the Example presented in this Section, is the cloning andcharacterization of a human homologue of the mouse C5 gene. The mouse C5gene is a homolog of the mouse brown fat uncoupling protein, and,further, as demonstrated, above, in Section 8, the gene isdifferentially expressed in obese versus lean littermate controls. Thus,C5 can represent a gene whose product is involved in body weightdisorders, and/or processes involved in body weight or appetiteregulation. Likewise, human C5 can also represent a gene whose productis involved in such disorders and/or processes in humans.

10.1. Materials and Methods

Human C5 was cloned from a human fetal spleen library (Stratagene). Theprobe used for the hybridization was a 0.9 kb partial cDNA clone of themouse C5 gene. Filters used for hybridization were NitroPlus 2000(Micron Separations, Inc.). Hybridization and washing conditions were asper manufacturer's instructions for low stringency hybridizations,except that the hybridization steps were carried out at 42° C.

Northern analysis was performed as described, above, in Section 7.1.

10.2. Results

The mouse C5 gene sequence was used, in conjunction with the methodstaught, above, in Sections 5.2.1 and 10.1, to isolate a cDNA clonecorresponding to human C5. The nucleotide sequence of the human C5 cDNAclone is shown in FIGS. 17A, 17B (bottom line; SEQ ID NO:38). The FIGS.17A, 17B nucleotide sequence (SEQ ID NO:38) encodes all but the firstten amino acids of the human C5 gene product.

The human C5 amino acid sequence derived from the cDNA sequence is alsodepicted in FIGS. 17A, 17B (top line; SEQ ID NO: 56). Using standardwell known techniques, the human C5 nucleotide sequence can be used toisolate the most 5' end of the C5 coding sequence. Alternatively, inorder to obtain a full length C5 protein, a nucleotide sequence encodingthe first 10 amino acids of the mouse C5 protein may be ligated to the5' end of the human cDNA sequence disclosed herein, such that an entirelinear C5 coding sequence is generated and a full length C5 protein canbe expressed using standard techniques. Further, the entire amino acidsequence of such a C5 protein can be synthesized by, again, utilizingstandard techniques well known to those of skill in the art.

The amino acid sequence of such a C5 protein is depicted in FIG. 18 (SEQID NO: 51). The first ten amino acid residues represent sequence derivedfrom the mouse C5 protein while the rest of the amino acid residuesrepresent sequence taken from the human C5 protein. Given the highdegree of amino acid conservation which exists between the mouse and thehuman C5 amino acid sequences, it is expected that the protein depictedin FIG. 18 represents a functional C5 protein.

A Northern analysis of the tissue distribution of human C5 mRNAtranscripts was performed, as depicted in FIG. 19. RNA from brain,heart, placenta, lung, liver, muscle, kidney, and pancreas tissues wereisolated and analyzed for C5 expression. As in the mouse, theapproximately 1.8 kb human C5 transcript exhibits a complex pattern oftissue distribution, with mRNA accumulation appearing to be greatest inmuscle tissue.

11. Deposit of Microorganisms

The following microorganisms were deposited with the AgriculturalResearch Service Culture Collection (NRRL), Peoria, Ill., on Aug. 23,1994 and assigned the indicated accession numbers:

    ______________________________________                                        Microorganism NRRL Accession No.                                              ______________________________________                                        famf049a      B-21318                                                         fahs005a      B-21320                                                         ______________________________________                                    

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 57                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 253 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGGCAGTACAACCAGATCCACTTTTATTAGGAACAAATACAATCTCAATCAGTACAAGTA60                GGCTTCAAGAGTTGATATTAATGGAAATCATCCAAATTACACTTGGGTCACAAATAATTA120               CCCCACATAAAAAGGGAAAAAAAAAATCTCATTCAGGGGAAGGGAAAGGTTTCCTGCAAT180               GGTTTTCATGGCAGTGGGTAGGTAGTCTTGCACTTTGGACTGGTCATATCTGTCAGTCTC240               TGGGCAGAGCAAA253                                                              (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 156 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CCTGAATGAGATTTTTTTTTTCCCTTTTTATGTGGGGTAATTATTTGTGACCCAAGTGTA60                ATTTGGATGATTTCCATTAATATCAACTCTTGAAGCCTACTTGTACTGATTGAGATTGTA120               TTTGTTCCTAATAAAAGTGGATCTGGTTGTACTGTC156                                       (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CAAAGTGCAAGACTACCTACCCACTGCCATGAAAACCATTGCAGGAAACCTTTCCCTTCC60                TGAATGAGATTTTTTTTTTCCCTTTTTATGTGGGG95                                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CCCTTCCAATACAAGAACTAAGTGGACTAGACTTCCAGTGATCCCTCTCCCAGCTCTTCC60                CTTTCCCAGTTGTCCCCACTGTAACTCAAAAG92                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CCCTTCCAATACAAGAACTAAGCAGACTAGACTTCCAGTGATCCCTCTCCCAGCTCTTCC60                CTCTCCCAGTTGTCCCCACTGTAACTCAAAGG92                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AAAGGATGGAATACCAAGGTCTTTTTATTCTTCGTGCCAAAAAAAAGA48                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 140 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATCTCTTTTGGCAGAACATGAATGCAGGTCACCTGGTGTCAATACTCAGCCAGGCTGAGA60                GCAACTTGGTGGCCTCGCTGGTTAAGGAGAGTGGTACTACAGCTTCCAATGTCTGGACTG120               GACTTCATGACCCTAAAAGT140                                                       (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 140 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AGCTCTTTTGCCAGAACATGAATGCAGGTCACCTGGTGTCAATACTCAGCCAGGCTGAGA60                GCAACTTTGTGGCCTCGCTGGTTAAGGAGAGTGGTACTACAGCTTCCAATGTCTGGACTG120               GACTTCATGACCCTAAAAGT140                                                       (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AACCGTCGTTGGCACTGGAGCAGTGGCTCCCTATTTCTCTTCAAGTCATGGGCCACTGGA60                GCTCCAAGCACTGCCAACCGTGGT84                                                    (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AACGCCTATGGTTCCTACTGTTATTATCTAATTGAAGACCGTTTGACCTGGGGGGAGGCT60                GATGT65                                                                       (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AGCATACAAAAAATGGAAGGACGAAAACTGTGAGGCACAGTACTCCTTTGTCTGCAAGTT60                CAGAGCCTAA70                                                                  (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 221 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGCTTAGTCAGCCACTTTACAGACTGGTCTTCCTGCTGGCACATACTGTGATGTCATCTC60                TGGAGATAAGGTCGATGGCAATTGCACTGGACTTAGAGTGAATGTTGGCAGTGATGGCAA120               AGCTCACTTTTCCATTAGTAACTCTGCTGAGGACCCATTTATTGCAATCCATGCTGACTC180               AAAATTGTAAGAATCTATATTAAAGAGATTTGGATTAGGAA221                                  (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 221 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGCTTTGTCAGCCACTTTACAGACTGGTCTTCCTGCTGGCACATACTGTGATGTCATCTC60                TGGAGATAAGGTCGATGGCAATTGCACTGGACTTAGAGTGAATGTTGGCAGTGATGGCAA120               AGCTCACTTTTCCATTAGTAACTCTGCTGAGGACCCATTTATTGCAATCCATGCTGACTC180               AAAATTGTAAGAATCTATATTAAAGAGATTTGGATTAAGCA221                                  (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 309 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AGAGTCAAGGGCTAGTGCGCACCGCAGCCAGCGCCCAGTACCGTGGCGTTCTGGGTACCA60                TCCTAACCATGGTGCGCACTGAGGGTCCACGCAGCCTCTACAATGGGCTGGTCGCCGGCC120               TGCAGCGCCAGATGAGCCTTGCCTCCGTCCGCATTGGCCTCTACGACTCTGTCAAACAGT180               TCTACACCAAGGGCTCAGAGCATGGAGGCATCGGGAGCCGCCTCCTGGCAGGTAGCACCA240               CAGGTGCCCTGGCCGTGGTTGTAGCCCAGCCTACAGATGTGGTAAAGGTCCGCTTCCAGG300               CTCCAGGCC309                                                                  (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 309 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AGATCCAAGGCGAGTTCCCGATCACCAGCGGCATCAGGTACAAAGGTGTCCTGGGGACAA60                TCACCACCCTGGCAAAAACGGAAGGGCCCCTGAAACTCTACAGCGGGTTGCCCGCCGGCC120               TCCAGAGACAAATCAGCTTCGCCTCGCTCAGGATCGGCCTCTACGACACGGTGCAGGAGT180               TCTTCACCTCGGGGGAAGAAACACCGAGTTTAGGAAGCAAGATCTCGGCCGGCCTAACAA240               CTGGAGGCGTGGCGGTGTTCATCGGGCAGCCCACAGAGGTCGTGAAAGTCAGGCTGCAAG300               CGCAGAGCC309                                                                  (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 814 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AAGATGCTGCTGCTGCTGCTGTGTTTGGGACTGACCCTAGTCTGTGTCCATGCAGAAGAA60                GCTAGTTCTACGGGAAGGAACTTTAATGTAGAAAAGATTAATGGGGAATGGCATACTATT120               ATCCTGGCCTCTGACAAAAGAGAAAAGATAGAAGATAATGGCAACTTTAGACTTTTTCTG180               GAGCAAATCCATGTCTTGGAGAAATCCTTAGTTCTTAAAATCCATCCTGTAAGAGATGAA240               GAGTGCTCCGAATTATCTATGGTTGCTGACAAAACAGAAAAGGCTGGTGAATATTCTGTG300               ACGTATGATGGATTCAATACATTTACTATACCTAAGACAGACTATGATAACTTTCTTATG360               GCTCATCTCATTAACGAAAAGGATGGGGAAACCTTCCAGCTGATGGGGCTCTATGGCCGA420               GAACCAGATTTGAGTTCAGACATCAAGGAAAGGTTTGCACAACTATGTGAGGAGCATGGA480               ATCCTTAGAGAAAATATCATTGACCTATCCAATGCCAATCGCTGCCTCCAGGCCCGAGAA540               TGAAGATTGGCCTGAGCCTCCAGTGTTGAGTGGAGACTTCTCACCAGGACTCCACCATCA600               TCCCTTCCTATCCATACAGCATCCCCAGTATAAATTCTGTGATCTGCATTCCATCCTGTC660               TCACTGAGAAGTCCAATTCCAGTCTATCCACATGTTACCTAGGATACCTCATCAAGAATC720               AAAGACTTCTTTAAATTTCTCTTTGATATACCCATGACAATTTTTCATGAATTTCTTCCT780               CTTCCTGTTCAATAAATGATTACCCTTGCACTTA814                                         (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 814 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ATGAAGATGCTGCTGCTGCTGTGTTTGGGACTGACCCTAGTCTGTGTCCATGCAGAAGAA60                GCTAGTTCTACGGGAAGGAACTTTAATGTAGAAAAGATTAATGGGGAATGGCATACTATT120               ATCCTGGCCTCTGACAAAAGAGAAAAGATAGAAGATAATGGCAACTTTAGACTTTTTCTG180               GAGCAAATCCATGTCTTGGAGAAATCCTTAGTTCTTAAATTCCATACTGTAAGAGATGAA240               GAGTGCTCCGAATTATCTATGGTTGCTGACAAAACAGAAAAGGCTGGTGAATATTCTGTG300               ACGTATGATGGATTCAATACATTTACTATACCTAAGACAGACTATGATAACTTTCTTATG360               GCTCATCTCATTAACGAAAAGGATGGGGAAACCTTCCAGCTGATGGGGCTCTATGGCCGA420               GAACCAGATTTGAGTTCAGACATCAAGGAAAGGTTTGCAAAACTATGTGAGGAGCATGGA480               ATCCTTAGAGAAAATATCATTGACCTATCCAATGCCAATCGCTGCCTCCAGGCCCGAGAA540               TGAAGAATGGCCTGAGCCTCCAGTGTTGAGTGGAGACTTCTCACCAGGACTCCACCATCA600               TCCCTTCCTATCCATACAGCATCCCCAGTATAAATTCTGTGATCTGCATTCCATCCTGTC660               TCACTGAGAAGTCCAATTCCAGTCTATCCACATGTTACCTAGGATACCTCATCAAGAATC720               AAAGACTTCTTTAAATTTCTCTTTGATATACCCATGACAATTTTTCATGAATTTCTTCCT780               CTTCCTGTTCAATAAATGATTACCCTTGCACTTA814                                         (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 277 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TGACCCGTACTTATTACAGCCGTACTGCTCCTATTATCACTACCAGTGCTAGCCGCAGGC60                ATTACTATACTACTAACAGACCGCAACCTAAACACAACTTTCTTTGATCCCGCTGGAGGA120               GGGGACCCAATTCTCTACCAGCATCTGTTCTGATTCTTTGGGCACCCAGAAGTTTATATT180               CTTATCCTCCCAGGATTTGGAATTATTTCACATGTAGTTACTTACTACTCCGGAAAAAAA240               GAACCTTTCGGCTATATAGGAATAGTATGAAAAAAAA277                                      (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 277 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TGATCCGTACTTATTACAGCCGTACTGCTCCTATTATCACTACCAGTGCTAGCCGCAGGC60                ATTACTATACTACTAACAGACCGCAACCTAAACACAACTTTCTTTGATCCCGCTGGAGGA120               GGGGACCCAATTCTCTACCAGCATCTGTTCTGATTCTTTGGGCACCCAGAAGTTTATATT180               CTTATCCTCCCAGGATTTGGAATTATTTCACATGTAGTTACTTACTACTCCGGAAAAAAA240               GAACCTTTCGGCTATATAGGAATAGTATGAGCAATAA277                                      (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 251 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TTGTTGCGATCCCACCAACCTACACTATGAGTTTCTTGTCCCGTTGATCCTGGGCTGCAT60                GAGGTTAAAGGGAATGATTGAGACCAGACAAGTCAGGGGTTGAAACTTAGAAAAGGTCAA120               AGGTACAGAAGAAACAGAGGACACTTCGTAGACTTGCAGAGGATATTTCAAAGGTAGCCA180               GAGAAGGGGGAAATTATACTATGTTGTCAATAGGAATAATAAAATAATAAAAGTAGATAT240               TATTTATGGAA251                                                                (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 251 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TTGTCACGATCCCACCAACCTACACTATGAGTTTCTTGTCCCGTTGATCCTGGGCTGCAT60                GAGGTTAAAGGGAATGATTGAGACCAGACAAGTCAGGGGTTGAAACTTAGAAAAGGTCAA120               AGGTACAGAAGAAACAGAGGACACTTCGTAGACTTGCAGAGGATATTTCAAAGGTAGCCA180               GAGAAGGGGGAAATTATACTATGTTGTCAATAGGAATAATAAAATAATAAAAGTAGATAT240               TATTTATGGCA251                                                                (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 226 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GGTKGTTGAGTGTGGCTGACTGGGATGCGCAGAGACCCAATGGTTCAGGCGCTGCCTGTC60                TGTCTGCCACTCCATCTTTCCTGTTGCCAGAGAGCCACCTGGCTGCCCCACCAGCCACCA120               TACCAAGGAGCATCTGGAGCCTCTTCTTATTTGGCCAGCACTCCCCATCCACCTGTCTTA180               ACACCACCAATGGCGTCCCCTTTCTGCTGAATAAATACATGCCCCC226                             (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 225 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GGGTGGTGAGTGTGGCTGACTGGGATGCGCAGAGACCCAATGGTTCAGGCGCTGCCTGTC60                TGTCTGCCACTCCATCTTTCCTGTTGCCAGAGAGCCACCTGGCTGCCCCACCAGCCACCT120               ACCAAGGAGCATCTGGAGCCTCTTCTTATTTGGCCAGCACTCCCCATCCACCTGTCTTAA180               CACCACCAATGGCGTCCCCTTTCTGCTGAATAAATACATGCCCCC225                              (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      CCGACCAATGCATTGACAACTGAATGGGTGGT32                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 155 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      TCCGCTCTGGATGCCAGGGTGATTCTGGGGGACCCCTCCACTGCATGGTGAACGGTCAGT60                ATGCTGTCCACGGAGTGACCAGCTTTGTGTCCAGCATGGGCTGTAATGTCGCCAGGAAGC120               CCACCGTCTTCACCAGAGTCTCTGCTTACATTTTC155                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 155 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      TCCGCTCTGGATGCCAGGGTGATTCTGGGGGACCCCTCCACTGCATGGTGAACGGTCAGT60                ATGCTGTCGACGGAGTGACCAGCTTTGTGTCCAGCATGGGCTGTAATGTCGCCAGGAAGC120               CCACCGTCTTCACCAGACTCTCTGCTTACATTTCC155                                        (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 82 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AGCCTTGGGCTCCATCCCTAATACTGCAACAGGAGCAGGGGAATGCTGCTGGTGTCTTGG60                TATCTGGGGCAAAGGTGGGGGG82                                                      (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GTGGGGGGTTAATGAAAAGCAACTCAGACTACTGAATCAGATACAGAAAGGCAAATAAAA60                ATCAATGTGTTA72                                                                (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 112 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GGGGCAGGGGATCTGCTCAGCTCTATGTTTGAGTTCAGTGAGAAGCTGAATGCCCTCCAG60                CTCAGTGATGAGGAAATGAGCTTGTTCACAGCAGTTGTTCTGGTATCTGCAG112                       (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 112 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GGAGCAGGGGATCTGCTCAGCTCTATGTTTGAGTTCAGTGAGAAGCTGAATGCCCTCCAG60                CTCAGTGATGAGGAAATGAGCTTGTTCACAGCAGTTGTTCTGGTATCTGCAG112                       (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GATCGATCTGGAATTGAAAATGTCAACTCAGTGGAGGCTTTGCAGGAAACACTCATCCGT60                GCACTAAGGACCTTAATAATGAAAAACCATCCAAATGAGGCCTCCATTTTTACAAAATT119                (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 83 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      AAAAACCATCCAAATGAGGCCTCCATTTTTACAAAATTACTTCTAAAGTTGCCAGATCTT60                CGATCTTTAAACAACATGCACTC83                                                     (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GGTAAGAAGTACAGTGTGGATGACCTGCACTCAATGGG38                                      (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 457 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CGCTGTCTCGGGAGGTCTGAAGACATGAAGCTGCTTCAGGTTCTCCTTGTTTTGCTGTTT60                GTGGCACTTGCAGATGGTGCACAGCCCAAAAGATGTTTTAGCAACGTAGAAGGCTACTGT120               AGGAAGAAATGCAGATTAGTGGAGATATCTGAGATGGGATGCCTGCATGGGAAATACTGT180               TGTGTTAATGAGCTGGAGAACAAAAAGCACAAGGAGCACTCAGTCGTTGAGGAGACAGTC240               AAACTCCAAGACAAGTCAAAAGTACAAGACTATATGATCCTGCCCACGGTCACATACTAC300               ACCATCACTATCTGAATGAACCACTTGTTCACGAAGGCCGTTGTCCCCTGCAGCCCCATG360               GAATCCAGTGGGCTGCTTCTGTCCTGTCTCTTTCCTTCTGTGAAACTTGAGTCTGCACAC420               AATAAAGTTCGACCCTTTTGGCTGAAAAAAAAAAAAA457                                      (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      MetLysLeuLeuGlnValLeuLeuValLeuLeuPheValAlaLeuAla                              151015                                                                        AspGlyAlaGlnProLysArgCysPheSerAsnValGluGlyTyrCys                              202530                                                                        ArgLysLysCysArgLeuValGluIleSerGluMetGlyCysLeuHis                              354045                                                                        GlyLysTyrCysCysValAsnGluLeuGluAsnLysLysHisLysGlu                              505560                                                                        HisSerValValGluGluThrValLysLeuGlnAspLysSerLysVal                              65707580                                                                      GlnAspTyrMetIleLeuProThrValThrTyrTyrThrIleSerIle                              859095                                                                        (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1205 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      ATGGTTGGTTTCAAGGCCACAGATGTGCCCCCAACAGCCACTGTGAAGTTCCTGGGGGCT60                GGGACAGCTGCCTGCATTGCAGATCTCATCACTTTCCCTCTGGATACCGCCAAGGTCCGG120               CTGCAGATCCAAGGGGAGAGTCAAGGGCTAGTGCGCACCGCAGCCAGCGCCCAGTACCGT180               GGCGTTCTGGGTACCATCCTAACCATGGTGCGCACTGAGGGTCCACGCAGCCTCTACAAT240               GGGCTGGTCGCCGGCCTGCAGCGCCAGATGAGCCTTGCCTCCGTCCGCATTGGCCTCTAC300               GACTCTGTCAAACAGTTCTACACCAAGGGCTCAGAGCATGGAGGCATCGGGAGCCGCCTC360               CTGGCAGGTAGCACCACAGGTGCCCTGGCCGTGGTTGTAGCCCAGCCTACAGATGTGGTA420               AAGGTCCGCTTCCAGGCTCCAGGCCGGGCTGGTGGTGGTCGGAGATACAGAGCACTGTCG480               AGCTACAAGAACATCACGAGAGGAGGGATCCGGGGCCTCTGGAAGGGACTCTCCCAATGT540               GCCCGTAATGCCATTGTCAACTGTGCTGAGCTGGTGACCTATGACCTCATCAAAGATACT600               CTCCTGAGCCACCTCATGACAGATGACCTCCCTTGCCACTTCACTTCTGCCTTCGGGGCG660               GGCTTCTGCACCACCGTCATCGCCTCCCCTGTGGATGTGGTCAAGACGAGATACATGACT720               CTGCTGGGCCAGTACCACAGCGCAGGTCACTGTGCCCTTACATGCTCGGAGGAGGGACCC780               GCGCTCTTCAACCAGGGGGTTATGCCTTCCTTTCTCCGCTTGGGATCCTGGAACGTAGTG840               ATGTTTGTCACCTATGAGCAGCTCCAAAGAGCCCTAATGGCTGCCTACCAATCTCGGGAG900               GCACCTTTCTGAGCCTCTCCATGCTGACCTGGACCCTGCTTCCCAGCCCTGCCCTGTCTT960               TTTCTTCATCCTCTGCCCAGTCCCATTCTCTTCCCATTTCCTGCACCCCGATTTACTTCC1020              CACCTCACCTCCCTGTGCCTCTGTACTGATGACTCACAGTGAGGAGGCCTGACACCAGAC1080              CCTGAGCCCTCAGCCCTTTCTACAGCTAAGCCCACATCTTCATCTTCATCCCCAGCCCAG1140              CCCAGCCCAGCTCAGCCAGCCTTCACCCATAAAGCAAGCTCAATGTTAAAAAAAAAAAAA1200              AAAAA1205                                                                     (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 303 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      MetValGlyPheLysAlaThrAspValProProThrAlaThrValLys                              151015                                                                        PheLeuGlyAlaGlyThrAlaAlaCysIleAlaAspLeuIleThrPhe                              202530                                                                        ProLeuAspThrAlaLysValArgLeuGlnIleGlnGlyGluSerGln                              354045                                                                        GlyLeuValArgThrAlaAlaSerAlaGlnTyrArgGlyValLeuGly                              505560                                                                        ThrIleLeuThrMetValArgThrGluGlyProArgSerLeuTyrAsn                              65707580                                                                      GlyLeuValAlaGlyLeuGlnArgGluMetSerLeuAlaSerValArg                              859095                                                                        IleGlyLeuTyrAspSerValLysGlnPheTyrThrLysGlySerGlu                              100105110                                                                     HisGlyGlyIleGlySerArgLeuLeuAlaGlySerThrThrGlyAla                              115120125                                                                     LeuAlaValValValAlaGlnProThrAspValValLysValArgPhe                              130135140                                                                     GlnAlaProGlyArgAlaGlyGlyGlyArgArgTyrArgAlaLeuSer                              145150155160                                                                  SerTyrLysAsnIleThrArgGlyGlyIleArgGlyLeuTrpLysGly                              165170175                                                                     LeuSerGlnCysAlaArgAsnAlaIleValAsnCysAlaGluLeuVal                              180185190                                                                     ThrTyrAspLeuIleLysAspThrLeuLeuSerHisLeuMetThrAsp                              195200205                                                                     AspLeuProCysHisPheThrSerAlaPheGlyAlaGlyPheCysThr                              210215220                                                                     ThrValIleAlaSerProValAspValValLysThrArgTyrMetThr                              225230235240                                                                  LeuLeuGlyGlnTyrHisSerAlaGlyHisCysAlaLeuThrCysSer                              245250255                                                                     GluGluGlyProAlaLeuPheAsnGlnGlyValMetProSerPheLeu                              260265270                                                                     ArgLeuGlySerTrpAsnValValMetPheValThrTyrGluGlnLeu                              275280285                                                                     GlnArgAlaLeuMetAlaAlaTyrGlnSerArgGluAlaProPhe                                 290295300                                                                     (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1255 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      CCTACTGCCACTGTGAAGTTTCTTGGGGCTGGCACAGCTGCCTGCATCGCAGATCTCATC60                ACCTTTCCTCTGGATACTGCTAAAGTCCGGTTACAGATCCAAGGAGAAAGTCAGGGGCCA120               GTGCGCGCTACAGTCAGCGCCCAGTACCGCGGTGTGATGGGCACCATTCTGACCATGGTG180               CGTACTGAGGGCCCCCGAAGCCTCTACAATGGGCTGGTTGCCGGCCTGCAGCGCCAAATG240               AGCTTTGCCTCTGTCCGCATCGGCCTGTATGATTCTGTCAAACAGTTCTACACCAAGGGC300               TCTGAGCATGCCAGCATTGGGAGCCGCCTCCTAGCAGGCAGCACCACAGGTGCCCTGGCT360               GTGGCTGTGGCCCAGCCCACGGATGTGGTAAAGGTCCGATTCCAAGCTCAGGCCCGGGCT420               GGAGGTGGTCGGAGATACCAAAGCACCGTCAATGCCTACAAGACCATTGCCCGAGAGGAA480               GGGTTCCGGGGCCTCTGGAAAGGGACCTCTCCCAATGTTGCTCGTAATGCCATTGTCAAC540               TGTGCTGAGCTGGTGACCTATGACCTCATCAAGGATGCCCTCCTGAAAGCCAACCTCATG600               ACAGATGACCTCCCTTGCCACTTCACTTCTGCCTTTGGGGCAGGCTTCTGCACCACTGTC660               ATCGCCTCCCCTGTAGACGTGGTCAAGACGAGATACATGAACTCTGCCCTGGGCCAGTAC720               AGTAGCGCTGGCCACTGTGCCCTTACCATGCTCCAGAAGGAGGGGCCCCGAGCCTTCTAC780               AAAGGGTTCATGCCCTCCTTTCTCCGCTTGGGTTCCTGGAACGTGGTGATGTTCGTCACC840               TATGAGCAGCTGAAACGAGCCCTCATGGCTGCCTGCACTTCCCGAGAGGCTCCCTTCTGA900               GCCTCTCCTGCTGCTGACCTGATCACCTCTGGCTTTGTCTCTAGCCGGGCCATGCTTTCC960               TTTTCTTCCTTCTTTCTCTTCCCTCCTTCCCTTCTCTCCTTCCCTCTTTCCCCACCTCTT1020              CCTTCCGCTCCTTTACCTACCACCTTCCCTCTTTCTACATTCTCATCTACTCATTGTCTC1080              AGTGCTGGTGGAGTTGACATTTGACAGTGTGGGAGGCCTCGTACCAGCCAGGATCCCAAG1140              CGTCCCGTCCCTTGGAAAGTTCAGCCAGAATCTTCGTCCTGCCCCCGACAGCCCAGCCTA1200              GCCCACTTGTCATCCATAAAGCAAGCTCAACCTTGAAAAAAAAAAAAAAAAAAAA1255                   (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      AAAGGATGGAATACCAAGGTCTTTTTATTCCTCGTGAAAAAAAAAAAA48                            (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      AACCGTCGNTGGCACTGGAGCAGTGGCTCCCTATTTCTCTTCAAGTCATGGGCCACTGGA60                GCTCCAAGCACTGCCAACCGGGTT84                                                    (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      AACGCCTATGGTTCCTACTGTTATTATCTAATTGAAGACCGCTTGACCTGGGGGGAGGCT60                GATCT65                                                                       (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      AGGATACAAAAAATGGGAGGACGAAAACTGTGAGGCACAGTACTCCTTGGTCTTGAAGTT60                CAGAGGCTAA70                                                                  (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      CCGACCAATGCATTGACAACTGAATGGGTKGT32                                            (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 82 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      AGCCTTGGGCTCCATCCCTAATACTGCAACAGGAGCAGGGGAATGCTGCTGGTGTCTTGG60                TATCTGGGGCAAAGGTGGGGGG82                                                      (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      GGGGGGGGTTCATGAAAAGCAACTCAGACTACTGAATCAGATACAGAAAGGCAAATAAAA60                ATCAATGTGTTA72                                                                (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      GATCGATCTGGAATTGAAAATGTCAACTCAGTGGAGGCTTTGCAGGAAACACTCATCCGT60                GCACTAAGGACCTTAATAATGRAAAAACCATCCAAATGAGGCCTCCATTTTTACAAAAT119                (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 83 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      AAAAACCATCCAAATGAGGCCTCCATTTTTACAAAATTACTTCTAAAGTTGCCAGRTCTT60                CGATCTTTAAACAACATGCACTC83                                                     (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GGTAAGAAGTACAGTGTGGATGACCTGCACTCAATGGG38                                      (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 160 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      GATCCCTGGCCAGAAGTACAAAGACACTAACCTGCTAATACTCTTTAAGGAAGATTACTT60                TGCAAAAAAAAATGAAGAAAGAAAGCAGAGCAAAGTGGAAGCTAAATTAAAAGCTAAACA120               AGAGCATGAAGGAAGACACAAGCCAGGAAGTACTGAAACC160                                   (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 160 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      GATCCCTGGCCAGAAGTACAAAGACACTAACCTGCTAATACTCTTTAAGGAAGATTACTT60                TGCAAAAAAAAATGAAGAAAGAAAGCAGAGCAAAGTGGAAGCTAAATTAAAAGCTAAACA120               AGAGCATGAAGGAAGACACAAGCCAGGAAGTACTGAAACC160                                   (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 309 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      MetValGlyPheLysAlaThrAspValProProThrAlaThrValLys                              151015                                                                        PheLeuGlyAlaGlyThrAlaAlaCysIleAlaAspLeuIleThrPhe                              202530                                                                        ProLeuAspThrAlaLysValArgLeuGlnIleGlnGlyGluSerGln                              354045                                                                        GlyProValArgAlaThrValSerAlaGlnTyrArgGlyValMetGly                              505560                                                                        ThrIleLeuThrMetValArgThrGluGlyProArgSerLeuTyrAsn                              65707580                                                                      GlyLeuValAlaGlyLeuGlnArgGlnMetSerPheAlaSerValArg                              859095                                                                        IleGlyLeuTyrAspSerValLysGlnPheTyrThrLysGlySerGlu                              100105110                                                                     HisAlaSerIleGlySerArgLeuLeuAlaGlySerThrThrGlyAla                              115120125                                                                     LeuAlaValAlaValAlaGlnProThrAspValValLysValArgPhe                              130135140                                                                     GlnAlaGlnAlaArgAlaGlyGlyGlyArgArgTyrGlnSerThrVal                              145150155160                                                                  AsnAlaTyrLysThrIleAlaArgGluGluGlyPheArgGlyLeuTrp                              165170175                                                                     LysGlyThrSerProAsnValAlaArgAsnAlaIleValAsnCysAla                              180185190                                                                     GluLeuValThrTyrAspLeuIleLysAspAlaLeuLeuLysAlaAsn                              195200205                                                                     LeuMetThrAspAspLeuProCysHisPheThrSerAlaPheGlyAla                              210215220                                                                     GlyPheCysThrThrValIleAlaSerProValAspValValLysThr                              225230235240                                                                  ArgTyrMetAsnSerAlaLeuGlyGlnTyrSerSerAlaGlyHisCys                              245250255                                                                     AlaLeuThrMetLeuGlnLysGluGlyProArgAlaPheTyrLysGly                              260265270                                                                     PheMetProSerPheLeuArgLeuGlySerTrpAsnValValMetPhe                              275280285                                                                     ValThrTyrGluGlnLeuLysArgAlaLeuMetAlaAlaCysThrSer                              290295300                                                                     ArgGluAlaProPhe                                                               305                                                                           (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      AGGGTCAGTGGTCTCCAGTCTTCTCCCAGTTTGGGCTTATTGATGTCCAATAAACAAGTT60                CTGTGTCTGCAAA73                                                               (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      AGGGGCAGGGGCTCCATTCTTCTCCCCGGTTTGGTCTGATTGATGTCCAATAAACAACTT60                CTGTATCTTCAAA73                                                               (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      CCCTACCAACTCTGTGCAGTGGTTCGCTAAAGGGTCAGTGGTCTCCAGTCTTCTCCCAGT60                TTGGG65                                                                       (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      CCTTACCAACTCTGTGCAGTGGCTCGCTAGCAGGGGCAGGGGCTCCATTCTTCTCCCCGG60                TTTGG65                                                                       (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 299 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      ProThrAlaThrValLysPheLeuGlyAlaGlyThrAlaAlaCysIle                              151015                                                                        AlaAspLeuIleThrPheProLeuAspThrAlaLysValArgLeuGln                              202530                                                                        IleGlnGlyGluSerGlnGlyProValArgAlaThrValSerAlaGln                              354045                                                                        TyrArgGlyValMetGlyThrIleLeuThrMetValArgThrGluGly                              505560                                                                        ProArgSerLeuTyrAsnGlyLeuValAlaGlyLeuGlnArgGlnMet                              65707580                                                                      SerPheAlaSerValArgIleGlyLeuTyrAspSerValLysGlnPhe                              859095                                                                        TyrThrLysGlySerGluHisAlaSerIleGlySerArgLeuLeuAla                              100105110                                                                     GlySerThrThrGlyAlaLeuAlaValAlaValAlaGlnProThrAsp                              115120125                                                                     ValValLysValArgPheGlnAlaGlnAlaArgAlaGlyGlyGlyArg                              130135140                                                                     ArgTyrGlnSerThrValAsnAlaTyrLysThrIleAlaArgGluGlu                              145150155160                                                                  GlyPheArgGlyLeuTrpLysGlyThrSerProAsnValAlaArgAsn                              165170175                                                                     AlaIleValAsnCysAlaGluLeuValThrTyrAspLeuIleLysAsp                              180185190                                                                     AlaLeuLeuLysAlaAsnLeuMetThrAspAspLeuProCysHisPhe                              195200205                                                                     ThrSerAlaPheGlyAlaGlyPheCysThrThrValIleAlaSerPro                              210215220                                                                     ValAspValValLysThrArgTyrMetAsnSerAlaLeuGlyGlnTyr                              225230235240                                                                  SerSerAlaGlyHisCysAlaLeuThrMetLeuGlnLysGluGlyPro                              245250255                                                                     ArgAlaPheTyrLysGlyPheMetProSerPheLeuArgLeuGlySer                              260265270                                                                     TrpAsnValValMetPheValThrTyrGluGlnLeuLysArgAlaLeu                              275280285                                                                     MetAlaAlaCysThrSerArgGluAlaProPhe                                             290295                                                                        (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1308 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GGAATTCGGCACGAGGAACTAGTCTCGAGTAGCTCTGTGTGTGTAGACACTCCCTAAGCC60                ACCCGAGGGCTGCCTCTGTGTGGTGGGGAGGTAGGAGAGGGGAAGAGGGTTGCTATTCTC120               ATTTTATACTTTTCCCACGGCTTCCAACTTTATGCCAATAACTCACAATTAAACAGGTCT180               CAGAACTAAGGGAGCTTTCAACACTTGGGCTAACTCAAGTGCAGTGAGCGAACGGGTTCT240               CCAAACCTGCTGGGTCCTGGCTACCTATGTCACACAGATGAGTGAGTCCACCTGCCTTCC300               ATTCTCTGAGCTTGTGGTCACAGCAGGAGTCTTCCAGGGCACTGCCTTGGSTTGGAAATG360               TAAGTCCGTCCGGAAATAGTATCCCCATCAGCTCTCTGGTGTTTGTGAGCTCTAGCATGC420               CATCCAGGTTCCAAGGAGGAGAGTCACGAAGGTCCCAGAGCCGCAAGGTGCACAGAGCAC480               CCTATAAACCCTGCAGTCCTCTTTGAGTCATGGGGAGGACTGTGGATCCCCTTCTTTGAG540               TCTAAAGAGGCTCAGGAATCCATATTCTCCACAAGGTCCAGGTGGGAAGTGCATGCATGA600               AATCAGACTCCAAAAAATGAACCACAAGTCACAGGAGAGAGAGAGAACTAGTCTCGAGCT660               CGTGCCGAATTCGGCACGAGGTGTAGCTCAGGTAGCCCAGGGAGCAATCAAGCTCTGCTT720               CCTGCTGCGCAGCTGTGGCTCGCTCCTGCCCGAACTGAGTCTCGCCGAGAGGACAGAGTT780               TGCTCACAAGATCTGGGACAAACTTCAGCAGTTAGGTGTCGTATATGATGTCAGTCATTA840               CAATGCTTTACTTAAAGTATATCTTCAAAATGAATACAAATTTTCACCTACTGACTTCCT900               GGCAAAGATGGAGGGAGCAAACATCCAACCAAATCGAGTAACATACCAGAGGCTGATAGC960               TGCCTACTGTAATGTTGGGGACATTGAAGGTGCCAGCAAGATCCTTGGATTTATGAAAAC1020              GAAAGACCTTCCGATCACAGAGGGCGTGTTCAGTGCTCTCGTCACAGGGCATGCGAGAGC1080              TGGGGATATGGAAAATGCAGAAAATATTCTCACAGTGATGAAACAGGCCGGCATTGAGCC1140              TGGCCCAGACACGTATCTGGCCTTGTTGAATGCACATGCTGAGAGGGGTGACATTGGCCA1200              GGTTAGGCAGATTCTGGAGAAAGTGGAGAAGTCAGACCATTACTTCATGGACCGCGACTT1260              CTTGCAGGTTATTTTTAGCTTCAGTAAGGCTGGCTACCCTCACTCGAG1308                          __________________________________________________________________________

What is claimed is:
 1. A method for diagnosing body weight disorders, comprising detecting, in a patient sample, the level of:(a) a C5 (SEQ ID NO.:36) or human C5 (SEQ ID NO.:38) gene transcript; (b) a gene transcript containing a nucleotide sequence present in a cDNA within E. coli clone fahs005a (NRRL Accession No. B-21320); (c) a gene transcript containing a nucleotide sequence which hybridizes under stringent conditions to the complement of the gene transcript of (a) or (b); (d) a gene transcript containing a nucleotide sequence which encodes an amino acid sequence depicted in SEQ ID NO.:37, SEQ ID NO.:56 or SEQ ID NO.:51; (e) a gene transcript containing a nucleotide sequence which encodes an amino acid sequence encoded by a cDNA within E. coli clone fahs005a (NRRL Accession No. B-21320); (f) a gene product containing an amino acid sequence encoded by a C5 (SEQ ID NO.:36) or human C5 (SEQ ID NO.:38) gene transcript; (g) a gene product encoded by a nucleotide sequence present in a cDNA within E. coli clone fahs005a (NRRL Accession No. B-21320); or (h) a gene product containing an amino acid sequence depicted in SEQ ID NO.:37, SEQ ID NO.:56 or SEQ ID NO.:51,so that if a differential level in the patient sample is detected relative to a corresponding non-body weight disorder sample, a body weight disorder is diagnosed.
 2. The method of claim 1 in which the level is induced in genetically obese individuals.
 3. The method of claim 1 in which the level is repressed in genetically obese individuals.
 4. The method of claim 1 in which the level is induced by fasting.
 5. The method of claim 1 in which the level is repressed by fasting.
 6. The method of claim 1 in which the level is repressed by refeeding of a fasted individual.
 7. The method of claim 1 in which the level is induced by refeeding of a fasted individual.
 8. The method of claim 1 in which the level is induced in underweight individuals.
 9. The method of claim 1 in which the level is repressed in underweight individuals.
 10. The method of claim 1 in which the level is induced in overweight individuals.
 11. The method of claim 1 in which the level is repressed in overweight individuals. 